Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

In vitro studies

Ames

Williams & Gatehouse, 1998 - Key Study: The test material showed no evidence of mutagenic activity in the Ames and liquid pre-incubation (Yahagi) tests, when tested up to a maximum concentration of 5086 µg test material per plate.

Lawlor 1993 - Supporting Study: The results of the Salmonella/Mammalian-Microsome Reverse Mutation Assay (Ames Test) indicate that under the conditions of this study, the test article did not cause a positive increase in the number of histidine revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9).

Chromosome Aberration in Human Whole Blood Lymphocytes

Murli 1996 - Key study: The test article was clastogenic in the in vitro whole blood human lymphocyte chromosomal aberration assay. A statistically significant increase in the frequency of cells with chromosomal aberrations was observed at the dose level of 3200 µg/mL in the presence of S9 metabolic activation. A statistically significant increase in the frequency of aberrant cells was also observed at 100 and 125 µg/mL in a single extended assay without S9 metabolic activation (50.3 hour treatment, 53.0 hour harvest).

Mouse Lymphoma

Allen 1998 - Key Study: The test material is considered to be a weak mutagen when tested using a 24-hour exposure regime. A dose-related increase in mutant frequency was observed. The highest dose tested, 250 µg/mL, produced a 6-fold increase in mutant frequency over the control.

In vivo studies

Mouse Micro-nucleus

Allen 1996 - Key Study: The test material induced a slight increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) at the highest dose, 1000 mg/kg/day, in male mice in the micronucleus assay; no increase was observed in females.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
18 September 1996 and 18 October
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
(Drug Approval and Licensing Procedures in Japan, 1992 ISBN 4-8407-1419-3 C3047.)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Male and female CD-1 mice were received from Charles River Breeding Laboratories, Inc. (Raleigh Facility) on 6 September 1995.
Upon receipt, animals were housed in suspended wire cages in the Toxicology Animal facility. All mice were fed Agway® Prolab® 3000 R-M-H Certified Pellets and were allowed tap water ad libitum.
All mice were acclimated to the controlled environment for 12 days prior to dosing.
Date of Birth: Males: 12 August 1995; Females: 8 August 1995
Body weight range: Male: 24.5 - 32.1g; female: 20.2 - 26.3g.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 71 +/- 2°F
- Humidity (%): 50 +/- 10%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark.
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: Methylcellulose
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 0.5%
- Amount of vehicle (if gavage or dermal): 10.0 mL/kg/day
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions of the appropriate concentrations of the test material for dosing were prepared prior to first dosing. Suspensions were stirred to give uniform mixing of the test compound. Calculations were entered in a Burroughs Wellcome official laboratory notebook or designated sheets.
Concentrations were verified by High Performance Liquid Chromatography at the beginning of the study.
Duration of treatment / exposure:
Three days
Frequency of treatment:
Three daily oral doses of the test material for groups 1-5 and 7-11 and a single oral dose for group 6 (positive control).
Post exposure period:
24 hours after final dose.
Remarks:
Doses / Concentrations:
Group 2
Basis:
analytical conc.
250 mg/kg/day
Remarks:
Doses / Concentrations:
Group 3
Basis:
analytical conc.
500 mg/kg/day
Remarks:
Doses / Concentrations:
Group 4
Basis:
analytical conc.
750 mg/kg/day
Remarks:
Doses / Concentrations:
Group 5
Basis:
analytical conc.
1000 mg/kg/day
No. of animals per sex per dose:
Five animals of each sex per dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: oral
- Doses / concentrations: 10.0 mL/kg at a concentration of 7.5 mg/mL
Tissues and cell types examined:
Bone marrow erythrocytes: polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)
Details of tissue and slide preparation:
Processing of Bone Marrow Cells and Preparation of Slides
The bone marrow cells were processed according to standard tcchniques for micronucleus analysis. Immediately following sacrifice, both femurs of each
mouse were exposed, separated from surrounding tissue, and rinsed free of any blood with Dulbecco's phosphate buffered saline. Two to three drops of a
solution of 50% fetal bovine serum in 1% (hypotonic) sodium citrate was dropped into the marrow canal using a #000 sable hair brush dipped in the solution.
The brush wetted with the same solution was then inserted into the marrow canal, and gently rotated to mix the marrow and to create a cell suspension. Four samples for each femur were streaked on each of two microscope slides. Slides from each animal were inscribed in duplicate with animal number, sex, group, and study number. Slides were air-dried for 1-4 days before fixing and staining.

Slide Staining
Slides were fixed in methanol for 5 minutes and allowed to air dry. Slides were then stained in 0.24 mM acridine orange in Hank's Balanced Salt Solution (pH 6.8) for approximately 3 minutes, rinsed in distilled or deionized water 3 times for 1-3 min each time, and air-dried. Slides were coverslipped before scoring using 1-2 drops Hank's Balanced Salt Solution.
Evaluation criteria:
The test article is assessed for its ability to produce an increased incidence of micronucleated polychromatic erythrocytes.
The final assessment of the test article is made by correlating the frequencies of micronucleated polychromatic erythrocytes to the doses inducing them.
Sex:
male
Genotoxicity:
positive
Remarks:
1000 mg/kg/day
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Antemortem Observations and Measurements
Clinical Signs
No animal deaths or clinical signs of toxicity were noted.

Body Weights
No treatment-related changes were noted.

Analysis of Micronuclei
Bone marrow was taken from groups 1-6. Since no treatment-related toxicity was observed, the 3 highest dose levels (500,750, and 1,000 mg/kg/day) and
the vehicle and positive control groups were analyzed. The % PCEs was based on analysis of 1000 erythrocytes per animal except in the vehicle control
and the test material-treated males where 2000 erythrocytes were scored. The number of micronucleated PCEs (MN-PCEs) was based on analysis of
2000 PCEs per animal except in the vehicle control and the test material-treated males where 4000 PCEs were scored. An additional 2000 PCEs were scored for males because of the high variability in the number of MN-PCEs observed in the highest dose group (1000 mg/kg/day). For the second scoring, additional slides which were not counted in the first scoring were used for most animals unless insufficient numbers of cells were present; all vehicle control and test material-treated groups for male mice were recoded prior to scoring. Data for the individual animals are shown in Tables 2 and 3 in Attachment 2.
Data are summarized by dose, sacrifice time and sex in Tables 4 (males) and 5 (females) (please see Attachment 2). The dose group average of MN-PCEs ±the standard deviation, normalized to 1000 PCEs, is given for each sex and sacrifice time.

For males only, there was a statistically significant difference between the group treated with 1000 mg/kg/day of test material and vehicle controls for the number of MN-PCEs. The mean number of MN-PCEs per 1000 PCEs was 3.9 ±1.96 in males dosed with 1,000 mg/kg/day as compared with 1.7 ± 1.28 for male vehicle control animals. For females there were no statistically significant differences between the test material-treated groups and vehicle controls for the number of MN-PCEs. For the percent PCEs [% PCEs = No. of PCEs x 100/(No. NCEs + PCEs)] there were no statistically significant differences between the test material-treated groups and vehicle controls.
For both sexes, there was a statistically significant (p < 0.05) increase in MN-PCEs of the positive control groups, cyclophosphamide (75 mg/kg) compared to the vehicle controls. The average numbers of MN-PCEs found in the male and female positive control animals were 36.1 and 54.6 per 1000 PCEs, respectively (Tables 4 and 5, please see Attachment 2). The vehicle control animals averaged 1.7 and 1.5 MN-PCEs per 1000 PCEs for males and females, respectively.
Mean percent PCE values for each animal are also summarized in Tables 4 and 5 (please see Attachment 2). Mean percent PCE values for vehicle control groups were 42.2 and 40.0
for males and females, respectively.
Conclusions:
Interpretation of results (migrated information): positive
The test material induced a slight increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) at the highest dose, 1000 mg/kg/day, in male mice in the micronucleus assay; no increase was observed in females.
Executive summary:

The test material induced a slight increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) at the highest dose, 1000 mg/kg/day, in male mice in the micronucleus assay; no increase was observed in females. One group of 5 male and 5 female mice was administered three daily oral doses of a vehicle control, and four groups of the same size received three daily oral doses of 250,500,750, or 1000 mg/kg of the test material. In addition, one group of 5 male and 5 female mice were dosed with a single oral dose of 75 mg/kg cyclophosphamide as the positive control. All mice were sacrificed at 24 hours after the final dose. Micronucleus analysis was performed on the three highest dose levels along with the vehicle and positive controls.

Under the conditions of this study, there was a slight, but statistically significant increase in the number of micronucleated PCEs observed in males treated with 1000 mg/kg/day of the test material as compared to the vehicle controls; the mean value for the control males was 1.7 micronucleated PCEs per 1000 PCEs and the corresponding value for males treated with 1000 mg/kg/day was 3.9. At lower doses of the test material in males and at all closes in females, there were no statistically significant differences between the test material-treated groups and vehicle controls. For the percent PCEs there were no statistically significant differences between the test material-treated groups and vehicle controls.

The mean plasma concentration for animals exposed to 1000 mg/kg/day was 42.0 and 72.1 µg/mL free base for males and females, respectively.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vivo:

A number of studies have been carried out on an analogue of the substance of interest to assess for genotoxicity. In total four in vitro studies and one in vivo study have been performed.

Of the in vitro studies, both Ames concluded that the test material was negative for mutagenicity however the remaining studies (including the in vivo) concluded that the test material should be considered to be mutagenic.

All of the studies were performed according to recognised guidelines and are therefore considered to be reliable. Based on this (with the exception of Lawlor (1993) which has been allocated as a supporting study to Williams & Gatehouse), all have been allocated as key studies of interest.

In vitro studies

Ames

Williams & Gatehouse, 1998 - Key Study

The test material was tested for microbial mutagenicity as part of a general screen to identify possible oncogenic and heritable genetic hazards that might be associated with the use of this compound. The test material was not mutagenic in the absence or presence of S9-mix towards any of the strains of Escherichia coli used in this study, when tested up to a concentration of 5086 µg test material per plate. Negative results were obtained in an earlier microbial mutagenicity screen with the Salmonella typhimurium tester strains (Lawlor, 1993).

All tests were carried out in the absence and presence of an in vitro metabolic activation system (rat liver S9-mix). The maximum test concentration used in both the plate incorporation (Ames) and liquid pre-incubation (Yahagi) tests was 5086 µg test material per plate.

Lawlor 1993 - Supporting Study

The test material was investigated for mutagenic activity in the Salmonella/Mammalian Microsome Reverse Mutation Assay (Ames Test). This assay evaluates the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimuriu mtester strains both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor-induced rat liver (S9).

The doses tested in the mutagenicity assay were selected based on the results of a dose range finding study using tester strain TA100 and ten doses of test article ranging from 5,000 to 6.67 µg per plate, one plate per dose, both in the presence and absence of S9.

The tester strains used in this study were TA98 , TA100, TA1535, TA1537 and TA1538. The assay was conducted using three plates per dose level both in the presence and absence of S9. Six doses of the test article were tested, from 5,000 to 100 µg per plate in both the presence and absence of S9.

Conclusions

The results of the Salmonella/Mammalian-Microsome Reverse Mutation Assay (Ames Test) indicate that under the conditions of this study, the test article did not cause a positive increase in the number of histidine revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9).

Chromosome Aberration in Human Whole Blood Lymphocytes

Murli 1996 - Key study

The test material was clastogenic in the in vitro whole blood human lymphocyte chromosomal aberration assay. A statistically significant increase in the frequency of cells with chromosomal aberrations was reproducibly observed in the presence of S9 metabolic activation. In addition, a statistically significant increase in the frequency of aberrant cells was also observed under certain conditions in the absence of S9 metabolic activation.

Data from a dose-ranging experiment showed that the test article caused a delay in the cell cycle at higher doses and subsequent experiments were conducted such that the harvest and fixation of the cells occurred at approximately 1.5 cell cycles or 1.5 cell cycles plus 24 hours. The highest test concentration was selected based on the effect of the test article on the pH of the tissue culture medium. The chromosome aberration assay was perfonned in two parts, an initial assay followed by a confirmatory assay.

For the initial assay, chromosome aberrations were examined in replicate cultures of cultured whole blood human lymphocytes (i) exposed to the test article for 27.1 hours at concentrations up to 125µg (free base)/mL without S9 metabolic activation, (ii) exposed for 3.0 hours at concentrations up to 3200µg (free base)/mL with and without S9 metabolic activation.

The confirmatory assay was performed with the same dose levels and slightly modified treatment time. In addition, an extended harvest time of 50.3 hours was added.

A minimum of one hundred metaphase cells were examined from each of two replicate cultures treated with the test material for a total of 200 cells per dose level.

In the initial assay, a statistically significant increase in the frequency of cells with chromosome aberrations was observed at the two highest dose levels in the 3.0 hour treatment in the presence and absence of S9 metabolic activation.

In the confirmatory assay, a statistically significant increase in the frequency of cells with chromosomal aberrations was observed at (i) the highest dose level for the 3.0 hour exposure in the presence of S9 metabolic activation, (ii) the two highest dose levels for the 50.3 hour exposure without S9 metabolic activation.

Positive controls, solvent controls and negative controls gave the expected results in all assays, showing that the system was capable of detecting chromosomal aberrations. No significant increase in percent polyploidy, hyperploidy, or endoreduplication was observed in the presence or absence of S9 metabolic activation.

Mouse Lymphoma

Allen 1998 - Key Study

The test material was tested for its ability to induce mutations at the heterozygous thymidine kinase (tk +/-) locus in the L5178Y/ tk +/- mouse lymphoma assay. The test material was not mutagenic when tested with 3-hour exposure in both the absence and in the presence of exogenous mammalian metabolic activation; however, the compound was a weak mutagen when tested using a 24-hour exposure regime in the absence of metabolic activation.

The test material was tested up to 1,000 µg/mL with a 3-hour exposure in the absence of S9 metabolic activation and up to 1,750 µg/mL in the presence of metabolic activation. At these concentrations, the relative total growth averaged 71% and 83%, in the absence and presence of S9 metabolic activation, respectively. Testing at higher concentrations was precluded by excessive pH changes.

In a 24-hour exposure in the absence of S9 metabolic activation, the test material was tested up to 250 µg/ mL; at this dose level the relative total growth of 12% and an increase in mutant frequency of 226 x 10-6over background was observed. At 125 µg/mL, the relative total growth was 81% with an increase of 57x 10-6over the background mutant frequency.

In vivo studies

Mouse Micro-nucleus

Allen 1996 - Key Study

The test material induced a slight increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) at the highest dose, 1000 mg/kg/day, in male mice in the micronucleus assay; no increase was observed in females. One group of 5 male and 5 female mice was administered three daily oral doses of a vehicle control, and four groups of the same size received three daily oral doses of 250,500,750, or 1000 mg/kg of the test material. In addition, one group of 5 male and 5 female mice were dosed with a single oral dose of 75 mg/kg cyclophosphamide as the positive control. All mice were sacrificed at 24 hours after the final dose. Micronucleus analysis was performed on the three highest dose levels along with the vehicle and positive controls.

Under the conditions of this study, there was a slight, but statistically significant increase in the number of micronucleated PCEs observed in males treated with 1000 mg/kg/day of the test material as compared to the vehicle controls; the mean value for the control males was 1.7 micronucleated PCEs per 1000 PCEs and the corresponding value for males treated with 1000 mg/kg/day was 3.9. At lower doses of the test material in males and at all closes in females, there were no statistically significant differences between the test material-treated groups and vehicle controls. For the percent PCEs there were no statistically significant differences between the test material-treated groups and vehicle controls.

The mean plasma concentration for animals exposed to 1000 mg/kg/day was 42.0 and 72.1 µg/mL free base for males and females, respectively.

Justification for selection of genetic toxicity endpoint

Mutagenic effects were noted in all but the two Ames studies. Furthermore as an in vivo study is available and its positive result is also repeated in two in vitro studies on the same material; this study is considered to be the study of interest for the purposes of classification and labelling.

Justification for classification or non-classification

Although all of the studies were carried out on an analogue material, the analogue is considered to be sufficiently similar to the substance of interest (please see attached data matrix and justification in Section 13 for additional details) for it to be used for the purposes of health and environment risk assessments.

Mutagenic effects were noted in all but the two Ames studies. Furthermore as an in vivo study is available and its positive result is also repeated in two in vitro studies on the same material; this study is considered to be the study of interest for the purposes of classification and labelling.

Under classification and labelling guidelines a test material should be considered to be classified as a Category 2 Mutagen when a positive result is indicated from an in vivo study carried out on somatic cells. As the analogue material shows a positive result in the in vivo Mouse Micornucleus study; and based on its similarities with the material of interest, the test material Abacavir Glutarate can be considered to be classified as a Category 2 Mutagen.