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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2018 to 04 February 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)
Deviations:
yes
Remarks:
Refer to main study report
GLP compliance:
yes
Type of assay:
transgenic rodent mutagenicity assay

Test material

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: Huntsman Advanced Materials; Batch Number AAF1453400- Expiration date of the lot/batch: 26 November 2021- Purity test date: Not providedSTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature, protected from light- Stability under test conditions: Stable under the conditions indicated above for the period of use- Solubility and stability of the test substance in the solvent/vehicle: Soluble in corn oil at concentrations of 10, 25, 60 and 75 mg/mLOxirane formulated in corn oil, at concentrations of 1.02 and 76.9 mg/mL, was determined to be stable for at least 72 hours when stored at 2-8 degreesC and for at least 24 hours when stored at room temperature.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Test substance formulations were stirred with a magnetic stir bar until homogeneous in appearance prior to use

Test animals

Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
Rats have been used historically in safety evaluation and genotoxicity studies and are recommended by regulatory agencies. Because this study was conducted in accordance with regulatory guidelines, alternatives could not be considered.The F344 (wild-type) strain is the background strain of the Big Blue rats, and is appropriate and acceptable for use in the 5-day range finder study. For dose range-finding studies, it is not necessary to use transgenic rodents of the same strain.The Big Blue in vivo mutation assay is a Transgenic Rodent (TGR) Mutation assay described in OECD Test Guideline 488 (2013). TGR assays in general and the Big Blue assay in particular have been reviewed by the OECD and identified in OECD Test Guideline 488 as being appropriate to investigate in vivo mutagenicity in any tissue of interest. The TGR assays are also recommended to investigate a potential mutagenic mode of action in the etiology of rodent tumors.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Source: In the 5-day range finder assay, wild-type Fischer 344 male rats (F344/NHsd) were received from Envigo, Frederick, MD. In the mutation assay, transgenic F344 Big Blue male rats were obtained from the BioReliance colony housed at Taconic Biosciences, Inc. (Germantown, NY). In addition, wild-type Fischer F344 male rats (F344/NHsd) were also received from Envigo, Frederick, MD for the TK cohort of the mutation assay. - Age at study initiation: Approximately 10 weeks old in the 5-day dose range finder assay; approximately 9 weeks old (mutation cohort) or approximately 8 weeks old (TK cohort) in the mutation assay - Weight at study initiation: 227.0 to 246.0 grams in the 5-day dose range finder assay; 188.1 to 232.5 grams (main study cohort) and 170.7 to 193.9 grams (TK cohort) in the mutation assay - Assigned to test groups randomly: [no/yes, under following basis: ] Yes, animals were randomized by body weight, using a computer-generated randomization program (Provantis Version 9.4.6.3), into dose groups. - Fasting period before study: None - Housing: Animals were housed in an environmentally-controlled room with continuous recording of room temperatures of 20.5 to 24.0 degreesC (approximately 69 to 75 degreesF) and a relative humidity of 30 to 70% with a 12-hour light/12-hour dark cycle (except when interrupted for study-related events). The animal rooms were supplied with at least 10 changes of HEPA-filtered air every hour. Animals for both the dose range-finder and mutation assays were multiple-housed during acclimation and following randomization, in polycarbonate cages. Sani-Chip hardwood bedding was used to absorb liquids. Cages and feeders were changed at least weekly. Animals were transferred to clean racks at least once every other week. Environmental enrichment was also provided in the form of Nylabones. - Diet (e.g. ad libitum): ad libitum, TEKLAD Global Diet #2018C (certified 18% protein rodent diet; Envigo, Madison, WI) - Water (e.g. ad libitum): ad libitum, via an automatic watering system (Napa Nectar gel packs were also provided, as needed) - Acclimation period: Animals were acclimated for 14 days in both the dose range-finder and mutation assays prior to the first dose administration. ENVIRONMENTAL CONDITIONS - Temperature (°C): 20.5 to 24.0 degreesC (approximately 69 to 75 degreesF) - Humidity (%): relative humidity of 30 to 70% - Air changes (per hr): The animal rooms were supplied with at least 10 changes of HEPA-filtered air every hour. - Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle (except when interrupted for study-related events) IN-LIFE DATES:5-day Dose Range FinderFrom: 15 May 2018 To: 02 June 2018Mutation AssayFrom: 12 June 2018 To: 23 July 2018 (TK cohort); 26 July 2018 (Mutation cohort)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil - Justification for choice of solvent/vehicle: Corn oil was selected as the vehicle of choice based on the Sponsor's request and compatibility with the test system. - Concentration of test material in vehicle: 250, 500 and 750 mg/kg/day in the dose range-finder; 100, 250, 600 and 750 mg/kg/day in the mutation assay - Amount of vehicle (if gavage or dermal): 10 mL/kg - Lot/batch no. (if required): MKCC0462 and MKCD1021
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of Oxirane was weighed into a suitable size amber glass vial, calibrated with a PTFE stir bar (no correction facter was used). Approximately 70% of the vehicle was added to the vial while stirring. After mixing, the remaining amount of vehicle was added to the QS line, and the formulations were again stirred until homogeneous in appearance. Formulations were prepared daily for the dose range-finder and at least every three days for the mutation assay.
Duration of treatment / exposure:
In the dose range-finder assay, animals received the vehicle or test substance formulations once daily by oral gavage for 5 consecutive days. In the mutation assay, animals received the vehicle or test substance formulations once daily by oral gavage for up to 28 consecutive days.Tissues from positive control animals from a previous BioReliance study (AE53KK.171.BTL) were used. In the previous study, animals received ENU in buffer solution (pH 6.00) at 20 mg/kg/day, on Days 1, 2, 3, 12, 19 and 26, at a dose volume of 10 mL/kg.
Frequency of treatment:
Once daily
Post exposure period:
In the dose range-finder assay, animals were sacrificed on Day 5. In the mutation assay, animals in the TK cohort were bled for bioanalytical evaluation on Day 28 and sacrificed after completion of blood collection. Animals in the mutation cohort were sacrificed on Day 31.
Doses / concentrations
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
Max dose indicated aboveAll doses in the mutation assay: 100.0, 250.0, 600.0, 750.0
No. of animals per sex per dose:
Dose range-finder: 3 animals/groupMutation assay: 6 animals/group (mutation cohort); 4 animals/group (TK cohort)
Control animals:
yes, concurrent vehicle
Positive control(s):
N-ethyl-N-nitrosourea - Justification for choice of positive control(s): ENU is a potent direct acting mutagen demonstrated to be mutagenic in the target tissues. The use of "packaging controls" is permitted by OECD Testing Guideline 488 as a way to reduce unnecessary animal usage. - Route of administration: Oral gavage - Doses / concentrations: Positive control animals from a previous BioReliance study (AE53KK.171.BTL) were used. In the previous study, animals received ENU in buffer solution (pH 6.00) at 20 mg/kg/day, on Days 1, 2, 3, 12, 19 and 26, at a dose volume of 10 mL/kg.

Examinations

Tissues and cell types examined:
The liver and glandular stomach were collected from animals in the vehicle and test substance-treated groups for cII mutant analysis. These tissues were also analyzed from animals in the positive control study.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose levels for the 5-day dose range-finder were selected in consultation with the Sponsor, based on toxicity data available to the Sponsor, and a limit dose of 1000 mg/kg/day. Dose levels were also selected with consideration to dose selection criteria discussed in ICH S2(R1) for repeat dose studies longer than 14 days and OECD Testing Guideline 407 for 28-day repeat dose toxicity studies.Dose levels for the mutation assay were selected based on the results of the 5-day dose range-finder. TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): In the dose range-finder assay, animals received the vehicle or test substance formulations once daily by oral gavage for 5 consecutive days and were sacrificed on Day 5. No necropsy was performed following scheduled sacrifice.In the mutation assay, animals received the vehicle or test substance formulations once daily by oral gavage for up to 28 consecutive days. Animals in the TK cohort were bled for bioanalytical evaluation on Day 28 and sacrificed after completion of blood collection. Animals in the mutation cohort were sacrificed on Day 31 and tissues were collected for cII mutant analysis. METHOD OF ANALYSIS: Liver and glandular stomach tissue samples were processed for DNA isolation from frozen tissues of the first 5 animals in the control group (Group 1) and three selected test substance-treated groups (Groups 2, 3 and 5 only; these groups were selected by the Study Director in consultation with the Sponsor). Tissues from the remaining animals/groups were retained frozen as reserve samples, if needed. Positive control tissues were extracted and DNA was used for packaging. Tissues were extracted following BioReliance SOPs, based on methods described for Agilent product RecoverEase for somatic tissues. Isolated DNA samples were stored at 2-8 degreesC.Isolated DNA was processed using Packaging Reaction Mix (PRM), purchased from New York University (New York, NY). This product is similar to Transpack manufactured by Agilent, Santa Clara, CA. PRM is used to isolate recoverable lambda shuttle DNA vectors from the genomic DNA and to package lambda shuttle vector DNA, using phage proteins and cofactors to create infectious lambda phage particles. Methods followed BioReliance SOPs, based on Agilent instruction manual titled "Lambda Select-cII Mutation Detection System for Big Blue Rodents" and the Agilent instruction manual titled "Transpack Packaging Extract for Lambda Transgenic Shuttle Vector Recovery".Packaged phage were incubated overnight at 37 +/- 1.0 degreesC, and then scored for plaque formation and titer determination; cII mutant selection plates were incubated for two days (between 40 to 48 hours) at 24 +/- 0.5 degreesC, and then scored for mutant plaques. At least 125,000 phage were evaluated from at least 2 packagings for each dose and tissue. Titer and mutant frequency were calculated per BIoReliance SOP.
Evaluation criteria:
The test substance was considered to have produced a positive response if it induces a statistically significant increase in the frequency of cII mutants in any dose level, outside the 95% control limits of the historical background mutant frequency range.The test substance was considered to have produced a negative response if no significant increase in cII mutant frequency was observed.Equivocal responses (if noted) were evaluated by the Study Director on a case-by-case basis, considering both statistical significance and biological relevance.Other criteria may have also been used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological relevance and plausibility, a qualitative dose-related increase). Any such considerations, if used, were clearly reported and described by the Study Director.
Statistics:
The incidence of all effects was analyzed separately by dose level. Dunnett's test was conducted on body weight, body weight changes and organ weight data. All statistics compared treated groups versus the concurrent control (Group 1) and were based on a significance value of p< 0.05.The individual animal is considered the experimental unit. Mutant frequency was calculated (number of mutant phage/number of total phage screened) for each tissue analyzed from each animal. Since this ratio is extremely small and may not be normally distributed, a log10 transformation of the MF data was performed.The statistical analysis of MF was conducted in two parts. Initially, the positive control group was compared to the vehicle control group. In the second part of the analysis, all treated groups (except the positive control) were compared to the vehicle control. In both instances, log10 transformed MF data was evaluated using 1-Way Analysis of Variance (ANOVA). The suitability of using the parametric ANOVA was confirmed by testing parameters of the log10 transformed MF data for normality and equal variance. If the data appeared normally distributed and exhibited equal variance, the parametric ANOVA analysis was used; if either test failed, a non-parametric method was used.Statistical analysis was performed using Minitab® 16.1.0.Further details are outlined in Appendix F of the report.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Dose-range finder: ruffled fur sporadically for one or two animals/group; Mutation asay (sporadically seen): hunched posture, piloerection for all animals (cage-side); hunched posture, ruffled fur for all animals (hands-on); some decreased motor activity.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY - Dose range: 250, 500, 750 mg/kg/day - Clinical signs of toxicity in test animals: During cage-side observations, ruffled fur was recorded for one or two animals/group, starting at 250 mg/kg/day, on Day 5 only. No other clinical signs were noted during cage-side or hands-on observations. RESULTS OF DEFINITIVE STUDY - Statistical evaluation: No biologically significant elevated MF was observed at the cII gene in the liver or glandular stomach of Big Blue male rats. The only statistically significant increase in MF (1.7-fold as compared to the vehicle control) was noted at 750 mg/kg/day for glandular stomach; however, the mean MF value at this dose level was slightly lower than the historical background value. Since individual MF values for all animals treated at 750 mg/kg/day were within the 95% CL of the historical background data, this result was not considered biologically significant.

Applicant's summary and conclusion

Conclusions:
Treatment with the test item at dose levels up to 750 mg/kg/day did not cause biologically significant increases in MF at the cII gene in the liver or glandular stomach of Big Blue® male rats.
Although a statistically significant increase in MF (1.7-fold as compared to the vehicle control) was noted at 750 mg/kg/day for glandular stomach, this value was lower than the historical background mean (30.3 x 10-6 vs. 34.2 x 10-6 respectively).
Executive summary:

This study investigated the effect of Oxirane on mutant frequency at the cII gene in liver and glandular stomach from male transgenic Fischer 344 Big Blue® rats; the study also included a 5day dose range-finding study. The Big Blue® Assay is a Transgenic Rodent (TGR) Mutation assay described in OECD Test Guideline 488 (OECD, 2013).

The 5-day range-finding study consisted of 12 wild-type Fischer 344 (F344) male rats (3/group). Group 1 animals received the vehicle (corn oil) and Groups 2 through 4 received Oxirane in the vehicle at doses of 250, 500, and 750 mg/kg/day, respectively, once daily by oral gavage for 5 consecutive days, at a dose volume of 10 mL/kg. Animals were sacrificed on Day 5; no necropsy was performed following scheduled sacrifice.

The mutation assay cohort consisted of a main cohort of 30 transgenic Fischer 344 Big Blue® male rats (6/group) and a TK cohort of 20 wild-type F344 male rats (4/group). Vehicle control animals (Group 1) were treated with corn oil. The test substance treated animals (Groups 2, 3, 4 and 5) were treated with Oxirane in the vehicle at doses of 100, 250, 600 and 750 mg/kg/day, respectively. Animals in Groups 1-5 were dosed once daily via oral gavage for up to 28 consecutive days. As part of a previous BioReliance study (AE53KK.171.BTL), six positive control animals for the Big Blue® assay (designated as Group 6) were treated with N-ethyl-N-nitrosourea (ENU) in buffer solution (pH 6.00) at 20 mg/kg/day, on Days 1, 2, 3, 12, 19, and 26. All doses were administered at a dose volume of 10 mL/kg.

Animals in the TK cohort were bled from the retro-orbital sinus (under 70% CO2/30% O2 anesthesia) on Day 28, for bioanalytical evaluation. After completion of blood collection, animals in this cohort were sacrificed by CO2 overdose and discarded without necropsy. Animals from the mutation assay cohort were sacrificed by CO2 overdose on Day 31. A partial necropsy was performed for animals in Groups 1-5; the liver and glandular stomach were collected for cII mutant analysis; the testes and duodenum were also collected but not analyzed for mutants. Collected tissues were weighed, flash frozen in liquid nitrogen, transferred to a freezer box on dry ice, and then stored in a freezer set at or below 60°C. Liver and glandular stomach tissue samples from the first five animals/group in Groups 1, 2, 3, 5 and 6 were processed for DNA isolation and analysis of cII mutants, following BioReliance SOPs.

No mortality was noted in this study. During the 5-day range-finding assay, a lack of weight gain was noted in the 750 mg/kg/day dose group. For the mutation assay cohort, decrease motor activity was noted occasionally in Oxirane-treated animals, at all dose levels. In addition, mean total body weight gains (Day 1-31) were significantly lower (37.2%) as compared to the vehicle control, at the 750 mg/kg/day dose level.

Treatment with Oxirane at dose levels up to 750 mg/kg/day did not cause biologically significant increases in mutant frequency (MF) at the cII gene in the liver or glandular stomach of Big Blue® male rats. Although a statistically significant increase in MF (1.7-fold as compared to the vehicle control) was noted at 750 mg/kg/day for glandular stomach, this value was slightly lower than the historical background mean (30.3 x 10-6 vs. 34.2 x 10-6 respectively). The positive control treatment with ENU produced statistically significant increases in MF for all tissues tested, demonstrating the utility of the test system to detect and quantify induced mutants, following exposure to a known direct acting mutagen. The study design and results obtained met protocol-specified assay acceptance criteria and were consistent with the study requirements of OECD TG 488 for transgenic rodent mutation assays.

A total of 27 samples were analyzed for the presence of glycidyl lauryl ether (GLE). Although several samples had detectable levels of GLE, no sample had results greater than the LLOQ of the method (2 μg/mL plasma). The initial LLOQ of the method was 1 μg/mL plasma, but due to the failure of the lowest calibration standard, the LLOQ of the method was raised to 2 μg/mL plasma.