Update to RIFM fragrance ingredient safety assessment, 2,4-dimethylbenzyl acetate, CAS Registry Number 62346-96-7.

The MOE is greater than 100. Without adjustment for specific uncertainty factors related to inter-species and intra-species variation, the material exposure by inhalation at 0.0040 mg/day is deemed to be safe under the most conservative consumer exposure scenario.

The RIFM approach to evaluating Natural Complex Substances (NCS).

The Research Institute for Fragrance Materials, Inc. (RIFM) has evaluated safety data for fragrance materials for 55 years. The safety assessment of Natural Complex Substances (NCS) is similar to that of discrete fragrance materials; all of the same endpoints are evaluated. A series of decision trees, reflecting advances in risk assessment approaches of mixtures and toxicological methodologies, follows a tiered approach for each endpoint using a 4-step process with testing only as a last resort: 1) evaluate available data on NCS; 2) verify whether the Threshold of Toxicological Concern (TTC) can be applied; 3) verify whether the NCS risk assessment can be achieved on a component basis; and 4) determine whether data must be generated. Using in silico tools, RIFM examined NCS similarities based on the plant part, processing, and composition of materials across 81 plant families to address data gaps. Data generated from the Creme RIFM Aggregate Exposure Model for over 900 fragrance NCS demonstrate that dermal exposure is the primary route of human exposure for NCS fragrance uses. Over a third of materials are below the most conservative TTC limits. This process aims to provide a comprehensive Safety Assessment of NCS used as a fragrance ingredient.

RIFM fragrance ingredient safety assessment, hydroxycitronellal dimethyl acetal, CAS Registry Number 141-92-4.

Hydroxycitronellal dimethyl acetal was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from read-across analog hydroxycitronellal diethyl acetal (CAS # 7779-94-4) show that hydroxycitronellal dimethyl acetal is not expected to be genotoxic. The repeated dose, reproductive, and local respiratory toxicity endpoints were evaluated using the TTC for a Cramer Class I material and the exposure to hydroxycitronellal dimethyl acetal is below the TTC (0.03 mg/kg/day, 0.03 mg/kg/day, and 1.4 mg/day, respectively). Data from hydroxycitronellal dimethyl acetal and from read-across material hydroxycitronellal diethyl acetal (CAS # 7779-94-4) show that there are no safety concerns for skin sensitization under the current declared levels of use. The phototoxicity/photoallergenicity endpoints were evaluated based on UV spectra; hydroxycitronellal dimethyl acetal is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; hydroxycitronellal dimethyl acetal was found not to be PBT as per the IFRA Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., PEC/PNEC), are <1.

RIFM fragrance ingredient safety assessment, p-tolyl acetate, CAS Registry Number 140-39-6.

The existing information supports the use of this material as described in this safety assessment. p-Tolyl acetate was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from read-across analog ethyl p-tolyl carbonate (CAS # 22719-81-9) show that p-tolyl acetate is not expected to be genotoxic. Data on read-across materials p-cresol (CAS # 106-44-5) and acetic acid (CAS # 64-19-7) provide a calculated MOE >100 for the repeated dose and reproductive toxicity endpoints. The skin sensitization endpoint was completed using DST for reactive materials (64 µg/cm2); exposure is below the DST. The phototoxicity/photoallergenicity endpoints were evaluated based on UV spectra; p-tolyl acetate is not expected to be phototoxic/photoallergenic. The local respiratory toxicity endpoint was evaluated using the TTC for a Cramer Class I material, and the exposure to p-tolyl acetate is below the TTC (1.4 mg/day).The environmental endpoints were evaluated; p-tolyl acetate was found not to be PBT as per the IFRA Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., PEC/PNEC), are <1.

RIFM fragrance ingredient safety assessment, 4-(p-hydroxyphenyl)-2-butanone, CAS Registry Number 5471-51-2.

The existing information supports the use of this material as described in this safety assessment. 4-(p-Hydroxyphenyl)-2-butanone was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data show that 4-(p-hydroxyphenyl)-2-butanone is not genotoxic. Data on 4-(p-hydroxyphenyl)-2-butanone provide a calculated MOE >100 for the repeated dose toxicity endpoint. The developmental and reproductive toxicity and local respiratory toxicity endpoints were evaluated using the TTC for a Cramer Class I material, and the exposure to 4-(p-hydroxyphenyl)-2-butanone is below the TTC (0.03 mg/kg/day and 1.4 mg/day, respectively). Data from 4-(p-hydroxyphenyl)-2-butanone show that there are no safety concerns for skin sensitization under the current declared levels of use. The phototoxicity/photoallergenicity endpoints were evaluated based on UV spectra; 4-(p-hydroxyphenyl)-2-butanone is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; 4-(p-hydroxyphenyl)-2-butanone was found not to be PBT as per the IFRA Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., PEC/PNEC), are <1.

RIFM fragrance ingredient safety assessment, isobutyl alcohol, CAS Registry Number 78-83-1.

The existing information supports the use of this material as described in this safety assessment. Isobutyl alcohol was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data show that isobutyl alcohol is not genotoxic. Data on isobutyl alcohol provide a calculated MOE >100 for the repeated dose toxicity and reproductive toxicity endpoints. Data from read-across material isoamyl alcohol (CAS # 123-51-3) show that there are no safety concerns for isobutyl alcohol for skin sensitization under the current declared levels of use. The phototoxicity/photoallergenicity endpoints were evaluated based on UV spectra; isobutyl alcohol is not expected to be phototoxic/photoallergenic. The local respiratory toxicity endpoint was evaluated using the TTC for a Cramer Class I material and the exposure to isobutyl alcohol is below the TTC (1.4 mg/day). The environmental endpoints were evaluated; isobutyl alcohol was found not to be PBT as per the IFRA Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., PEC/PNEC), are <1.

RIFM fragrance ingredient safety assessment, methyl 2-octynoate, CAS Registry Number 111-12-6.

Methyl 2-octynoate was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data show that methyl 2-octynoate is not genotoxic. Data provided methyl 2-octynoate a NESIL of 110 µg/cm2 for the skin sensitization endpoint. The repeated dose, developmental and reproductive, and local respiratory toxicity endpoints were evaluated using the TTC for a Cramer Class II material, and the exposure to methyl 2-octynoate is below the TTC (0.009 mg/kg/day, 0.009 mg/kg/day, and 0.47 mg/day, respectively). The phototoxicity/photoallergenicity endpoints were evaluated based on UV spectra; methyl 2-octynoate is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; methyl 2-octynoate was found not to be PBT as per the IFRA Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., PEC/PNEC), are <1.

RIFM fragrance ingredient safety assessment, methyl ionone (mixture of isomers), CAS registry number 1335-46-2.

Methyl ionone (mixture of isomers) was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from methyl ionone (mixture of isomers) show that the material is not genotoxic and provided a NESIL of 70,000 µg/cm2 for the skin sensitization endpoint. Data provided a calculated MOE >100 for the repeated dose toxicity and developmental toxicity endpoints, and data from read-across material (E)-ß-ionone (CAS # 79-77-6) provided a calculated MOE >100 for the reproductive toxicity endpoint. For the local respiratory endpoint, a calculated MOE >100 was provided by the read-across material ß-ionone (CAS # 14901-07-6). The phototoxicity/photoallergenicity endpoints were evaluated based on data and UV spectra; the material is not phototoxic/photoallergenic. The environmental endpoints were evaluated with data from the target chemical and read-across material α-allylionone (CAS # 79-78-7), and the material was not found to be PBT; its risk quotients, based on current volume of use in Europe and North America (PEC/PNEC), are <1.

RIFM fragrance ingredient safety assessment, methyl 2-nonenoate, CAS Registry Number 111-79-5.

The existing information supports the use of this material as described in this safety assessment. Methyl 2-nonenoate was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from read-across analog ethyl trans-2,cis-4-decadienoate (CAS # 3025-30-7) show that methyl 2-nonenoate is not expected to be genotoxic. The repeated dose, reproductive, and local respiratory toxicity endpoints were evaluated using the TTC for a Cramer Class I material, and the exposure to methyl 2-nonenoate is below the TTC (0.03 mg/kg/day, 0.03 mg/kg/day, and 1.4 mg/day, respectively). Data from the target and read-across analog isobutyl-2-butenoate (CAS # 589-66-2) do not indicate the material is a sensitizer. The phototoxicity/photoallergenicity endpoints were evaluated based on data and UV spectra; methyl 2-nonenoate is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; methyl 2-nonenoate was found not to be PBT as per the IFRA Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., PEC/PNEC), are <1.

RIFM fragrance ingredient safety assessment, isobutyl propionate, CAS Registry Number 540-42-1.

The existing information supports the use of this material as described in this safety assessment. Isobutyl propionate was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from read-across analog isobutyl acetate (CAS # 110-19-0) show that isobutyl propionate is not expected to be genotoxic. Data from read-across analog isoamyl acetate (CAS # 123-92-2) show that there are no safety concerns for isobutyl propionate for skin sensitization under the current declared levels of use. The repeated dose and reproductive endpoints were evaluated using the TTC for a Cramer Class I material, and the exposure to isobutyl propionate is below the TTC (0.03 mg/kg/day and 0.03 mg/kg/day, respectively). For the local respiratory endpoint, a calculated MOE >100 was provided by read-across analog butyl acetate (CAS # 123-86-4). The phototoxicity/photoallergenicity endpoints were evaluated based on UV spectra; isobutyl propionate is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; isobutyl propionate is not PBT as per the IFRA Environmental Standards. For the risk assessment, isobutyl propionate was not able to be risk screened as there were no reported volumes of use for North America or Europe in the 2015 IFRA Survey.

RIFM fragrance ingredient safety assessment, propanal diethyl acetal, CAS registry number 4744-08-5.

There are insufficient toxicity data on the target material propanal diethyl acetal (CAS # 4744-08-5). Hence, in silico evaluation was conducted to determine read-across analogs for this material. Based on structural similarity, reactivity, metabolism data, physical-chemical properties, and expert judgment, analogs acetal (CAS # 105-57-7) and butane, 1,1'-[methylenebis(oxy)]bis- (CAS # 2568-90-3) were identified as read-across materials with sufficient data for toxicological evaluation of genotoxicity.

RIFM fragrance ingredient safety assessment, 2-ethyl-1-hexanol, CAS registry number 104-76-7.

The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity, skin sensitization, as well as environmental safety. Data show that this material is not genotoxic. Data from the suitable read across analog 2-butyloctan-1-ol (CAS # 3913-02-8) show that this material does not have skin sensitization potential. The reproductive and local respiratory toxicity endpoints were completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class I material (0.03 and 1.4 mg/day, respectively). The developmental and repeat dose toxicity endpoints were completed data on the target material which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework.

RIFM fragrance ingredient safety assessment, linalyl benzoate, CAS Registry Number 126-64-7.

The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data show that this material is not genotoxic. Data from the suitable read across analog linalyl phenylacetate (CAS # 7143-69-3) show that this material does not have skin sensitization potential. The repeated dose toxicity endpoint was completed using linalyl cinnamate (CAS # 78-37-5) as a suitable read across analog, which provided a MOE > 100. The developmental and reproductive toxicity endpoint was completed using linalool (CAS # 78-70-6), dehydrolinalool (CAS # 29171-20-8), benzoic acid (CAS # 65-85-0) and sodium benzoate (CAS # 532-32-1) as suitable read across analogs, which provided a MOE > 100. The local respiratory toxicity endpoint was completed using linalool (CAS # 78-70-6) and benzoic acid (CAS # 65-85-0) as suitable read across analogs, which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework along with data from the suitable read across analog linalyl cinnamate (CAS # 78-375).

RIFM fragrance ingredient safety assessment, linalyl cinnamate, CAS Registry Number 78-37-5.

The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data show that this material is not genotoxic nor does it have skin sensitization potential. The reproductive and local respiratory toxicity endpoints were completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class I material (0.03 and 1.4 mg/day, respectively). The developmental toxicity endpoint was completed using linalool (CAS # 78-70-6), dehydrolinalool (CAS # 29171-20-8) and cinnamic acid (CAS # 621-82-9) as suitable read across analogs, which provided a MOE > 100. The repeated dose toxicity endpoint was completed using data on the target material which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework.

RIFM fragrance ingredient safety assessment, 2-methylundecanol, CAS Registry Number 10522-26-6.

This material was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data from the suitable read across analogs 2-butyloctan-1-ol (CAS # 3913-02-8) and 2-ethyl-1-hexanol (CAS # 104-76-7) show that this material is not genotoxic nor does it have skin sensitization potential. The reproductive and local respiratory toxicity endpoints were completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class I material (0.03 and 1.4 mg/day, respectively). The repeated dose toxicity endpoint was completed using 2-ethyl-1-hexanol (CAS # 104-76-7) and 1-heptanol, 2-propyl (CAS # 10042-59-8) as suitable read across analogs, which provided a MOE > 100. The developmental toxicity endpoint was completed using 2-ethyl-1-hexanol (CAS # 104-76-7) as a suitable read across analog, which provided a MOE > 100 The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework.

RIFM fragrance ingredient safety assessment, Isopulegol, CAS Registry Number 89-79-2.

This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data show that this material is not genotoxic nor does it have skin sensitization potential. The repeated dose, developmental and reproductive, and local respiratory toxicity endpoints were completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class I material (0.03, 0.03 mg/kg/day and 1.4 mg/day, respectively). The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework.

Expression and characterization of four recombinant human dihydrodiol dehydrogenase isoforms: oxidation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene to the activated o-quinone metabolite benzo[a]pyrene-7,8-dione.

The bioactivation of polycyclic aromatic hydrocarbons (PAHs) to their ultimate carcinogenic forms proceeds via the formation of proximate carcinogen trans-dihydrodiols. Previous studies demonstrated that rat liver 3 alpha-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3 alpha-HSD/DD), a member of the aldo-keto reductase (AKR) superfamily, oxidizes PAH trans-dihydrodiols to redox-cycling o-quinones. Multiple closely related AKRs exist in human liver; however, it is unclear which, if any, participate in PAH activation by catalyzing the NADP+ -dependent oxidation of PAH trans-dihydrodiols. In this study, cDNAs encoding four human DD isoforms were isolated from HepG2 cells using isoform-selective RT-PCR. The recombinant proteins were overexpressed in Escherichia coli, purified to homogeneity, and kinetically characterized. Calculation of KM and kcat values of each isoform for model substrates revealed that they possessed enzymatic activities assigned to native human liver DD1, DD2, DD4, and type 2 3alpha-HSD (DDX) proteins. The ability of human DDs to oxidize the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene (BP-diol) was then examined. A reverse phase HPLC radiochemical assay demonstrated that all four isoforms oxidize (+/-)-BP-diol in the following rank order: DD2 > DD1 > DD4 > DDX. Each DD consumed the entire racemic BP-diol mixture, indicating that both the minor (+)-S,S- and major (-)-R,R-stereoisomers formed in vivo are substrates. First-order decay plots showed that DD1 and DD2 displayed preferences for one of the stereoisomers, and circular dichroism spectroscopy indicated that this isomer was the (+)-7S, 8S-enantiomer. The products of these reactions were trapped as either glycine or thiol ether conjugates of benzo[a]pyrene-7,8-dione (BPQ), indicating that the initial oxidation product was the reactive BPQ. Thus, human liver possesses multiple AKRs which contribute to PAH activation by catalyzing the NADP+-dependent oxidation of PAH trans-dihydrodiols to redox-active o-quinones.

Overexpression and mutagenesis of the cDNA for rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase. Role of cysteines and tyrosines in catalysis.

The overexpression and purification of recombinant rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD; EC 1.1.1.50) in Escherichia coli are described. The properties of the homogeneous recombinant 3 alpha-HSD (r3 alpha-HSD) confirm that a single polypeptide can function as a HSD, as a dihydrodiol dehydrogenase, and as an aromatic aldehyde, ketone, and quinone reductase. Cys-170, Cys-242, and Cys-217, implicated by bromoacetoxysteroid affinity-labeling agents as points of contact for the C-3, C-11, and C-17 positions of steroid ligands, were mutated to alanines. Unexpectedly, the homogeneous C170A and C242A mutants were kinetically similar to wild-type r3 alpha-HSD. By contrast, the C217A mutant gave Km values that were 4-fold higher for androstanedione and 2-fold higher for NADH. Inspection of the recently solved crystal structure of rat liver 3 alpha-HSD (Hoog, S. S., Pawlowski, J. E., Alzari, P. M., Penning, T. M., and Lewis, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2517-2521) places Cys-170 and Cys-242 on the periphery of an alpha/beta-barrel so that they cannot be involved in catalysis of steroid recognition. This demonstrates that bromoacetoxysteroid affinity-labeling agents may provide misleading information regarding the topography of steroid hormone binding sites. When NADPH was modeled into the crystal structure of 3 alpha-HSD, Tyr-55 was implicated as the general acid, since it is in close proximity to the C-4 position of the nicotinamide ring and could polarize the substrate carbonyl. In support of this model, the purified Y55F mutant was found to be catalytically inactive, but still formed an E-NADPH complex (measured by fluorescence titration) and an E-NADH-testosterone complex (measured by equilibrium dialysis). The ability of the Y55F mutant to form binary and ternary complexes, but not aid in hydride transfer, is consistent with Tyr-55 acting as the general acid. 3 alpha-HSD is a member of the aldo-keto reductase superfamily, and Tyr-55 is invariant in members of this family where it may perform a similar function. Tyr-205 is present in a pentapeptide sequence that is conserved in HSDs that belong to the short-chain alcohol dehydrogenase family and has been implicated as the general acid within these enzymes. The Y205F mutant was found to be kinetically similar to wild-type r3 alpha-HSD.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of 5 beta-dihydrocortisone reduction in rat liver cytosol: a rapid spectrophotometric screen for nonsteroidal anti-inflammatory drug potency.

The purified NAD(P)-linked 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol is powerfully inhibited by the nonsteroidal anti-inflammatory drugs in rank-order of their therapeutic potency. This observation has now been developed into a rapid screen for predicting the potency of these agents. The screening method requires the preparation of a rat liver cytosol and access to a recording spectrophotometer. By monitoring the NADPH-linked reduction of 5 beta-dihydrocortisone catalyzed by these cytosols in the presence of nonsteroidal anti-inflammatory drugs, a high correlation between their log IC50 values for this reaction with their recommended daily dose in humans can be obtained (r = 0.960; n = 10; p greater than 0.0001). This correlation scans four orders of magnitude and demonstrates the value of this method for screening the potency of these drugs. This method is superior to traditional predictive methods (e.g., measurement of cyclooxygenase inhibition), since this assay measures the change in absorbance of a pyridine nucleotide over 5 min and is considerably more rapid than the extraction, separation, and quantitation of prostaglandins. The method does not require enzyme purification, and the cytosol, which can be prepared in a matter of hours, can be stored at -80 degrees C and used routinely for screening purposes.