Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Toluene has been examined for mutagenicity both in vitro and in vivo in a range of recognised core assay types. It has shown negative results for mutagenicity both in vitro and in vivo. It is concluded that the available data are sufficient and indicate that toluene has no significant genotoxicity

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Near guideline study. GLP status unknown, Limitations in design and reporting but otherwise adequate for assessment.
Principles of method if other than guideline:
Rats were dosed with test compound on both an acute and a subchronic schedule. Acute study rats were killed at 6, 24 and 48 hours after treatment, subchronic study rats were killed 6 hours after last dose. Bone marrow spreads were prepared. The spreads were assessed for mitotically active cells that had been arrested at metaphase using colchicine, for structural changes and rearrangements of their chromosomes.
GLP compliance:
not specified
Type of assay:
other: rat bone marrow cytogenetic analysis
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Duration of treatment / exposure:
single dose (acute study); 5 days (subchronic study)
Frequency of treatment:
single dose (acute study), once per day, 24 hours apart (subchronic study)
Post exposure period:
6, 24 and 48 hours after dose (acute study), 6 hours after final dose (subchronic study)
Remarks:
Doses / Concentrations:
0.025, 0.082, 0.247 mL/kg
Basis:
other: nominal in DMSO
No. of animals per sex per dose:
5 per dose level (no information on sex of animals)
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: ip (assumed)
- Doses / concentrations: 0.3 mg/kg
Tissues and cell types examined:
rat bone marrow
Evaluation criteria:
Evaluated for presence of chromosome aberrations. Breaks, gaps, fragments and chromosome rearrangement recorded. Wherever possible, 50 cells were located and scored per slide.
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
No evidence of genotoxicity
Executive summary:

An evaluation of the in vivo clastogenicity of toluene was investigated in rat bone marrow. Toluene was administrated i.p. to the animals at 0.025 mL/kg, 0.082 mL/kg and 0.247 mL/kg corresponding to 22, 71, and 215 mg/kg body weight. The study was negative, as all chromosomal aberrations observed were in the range of the spontaneous background. No depression of the mitotic index was observed at any of the dose levels tested.

Toluene is not genotoxic in mammalian cells in vivo.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

LOA is currently reviewing the human and animal data supporting Human Health for Toluene. It is expected to be completed by Q4 2020.

Additional information from genetic toxicity in vivo:

The genotoxic potential of toluene was reviewed and reported in the EU RAR (2003). No additional relevant data have been found in the updated literature review.

Non-human information

In vitro data

The key studies are considered to be bacterial mutation assays (Bos et al, 1981; Haworth et al, 1983) and mammalian cell gene mutation assays (API, 1978; McGregor et al, 1988). These are two recognised core assay types for investigating mutagenicity in vitro.

Haworth et al (1983) examined toluene in the Ames test, in S typhimurium strains TA1535, TA1537, TA98, TA100, using a pre-incubation protocol, which provides a more stringent test for volatile materials. Dose-levels up to 1000 µg per plate were used, with the top dose being limited by toxicity to the bacteria. Toluene was negative both without auxiliary metabolic activation (S9) and also with S9 from rats or Syrian hamsters pre-treated with Aroclor 1254. Bos et al (1981) examined toluene in the standard Ames test using S. typhimurium TA1535, TA1537, TA1538, TA98, TA100, both in the absence of S9 and in the presence of S9 from untreated rats and rats pre-treated with Aroclor. Doses of up to 2000 µg/plate were used and toluene was again negative in the assay.

API (1978) examined toluene in the L5178Y (TK+/-) mouse lymphoma assay at doses of 0.05, 0.1, 0.15, 0.2 and 0.3 µL/mL, with greater than 90% reduction in cell survival seen at the top dose level (equivalent to 260 µg/mL). Toluene was negative in the assay. McGregor et al (1988) also tested toluene in the L5178Y (TK+/-) mouse lymphoma assay both with and without S9 from Aroclor induced rats. Doses of up to 500µg/mL were used, but at the highest doses, total lethality was observed. Although increases in mutant frequency were reported, these were <2 times the control value in three of the four experiments conducted. The authors concluded the results of their study as questionable. Such small increases in mutant frequency are of limited biological significance, and the study is considered to have shown no evidence of significant genotoxic activity for toluene.

There are a number of additional reports of toluene being found negative using in vitro assays, including endpoints of gene mutation and DNA repair in bacteria, gene conversion in yeast (EU RAR, 2003), and in mammalian cells with endpoints of DNA strand breaks, DNA damage/repair, sister chromatid exchange and micronucleus induction (EU RAR, 2003). The data from these assays all support the above conclusion that toluene is not mutagenic in vitro and indicate that further studies are not required (EU RAR, 2003).

In vivo data

The key study is considered to be a cytogenetic study in the rat (API, 1978). This is a recognised core assay type for investigating mutation in vivo.

Toluene was administrated intraperitoneally to rats at 22, 71, and 215 mg/kg and the bone marrow examined for chromosomal aberrations. Toluene gave a negative response. A number of additional cytogenetic studies have been carried out in the rat and mouse, and their findings support the conclusion that toluene is non-genotoxic in somatic cells in vivo (EU RAR, 2003).

In addition to somatic cells, toluene has been examined for genotoxicity in germ cells. API (1981) evaluated toluene in the dominant lethal assay in male CD-1 mice. Toluene was administered at 100 and 400 ppm by inhalation, for 6 hours per day, 5 days per week for 8 weeks. Following treatment, the animals were mated sequentially to two females per week for each of 2 weeks. Toluene was considered not to be mutagenic as it did not increase pre- or post implantation embryo loss.

Human information

A number of studies have examined occupational settings where toluene has been in use, and reported changes in certain genotoxicity parameters. However, equivocal results have been obtained and the inability to control confounding factors such as possible co-exposure to other agents does not allow meaningful conclusions for toluene (EU RAR, 2003). Prolonged exposure to 50 ppm toluene did not induce increased frequencies of sister chromatid exchange in peripheral blood lymphocytes of human volunteers (EU RAR, 2003).


Justification for selection of genetic toxicity endpoint
Available data indicate that the toluene has no significant genotoxicity in bacterial and mammalian systems in vitro and/or in vivo

Justification for classification or non-classification

Toluene has shown negative results for mutagenicity both in vitro and in vivo in a range of recognised core assay types. It is concluded that the available data are sufficient and that no classification under GHS / CLP is warranted.