Reaction products of phosphoryl trichloride and 2-methyloxirane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471 (Safepharm Laboratories Ltd, 1992): Negative

OECD 476 (Covance Laboratories Ltd, 2005): Negative without S9 / Positve with S9 (see corresponding in vivo study - Covance Laboratories Ltd, 2006)

in vitro cytogenicity / micronucleus: An in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not recorded
- Periodically "cleansed" against high spontaneous background: not recorded

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Initially examined both in the absence and presence of Aroclor 1254 induced rat liver S9 fraction at doses of 62.5, 125, 250, 500, 1000 and 2000 µg/ml. Complete toxicity was observed at 500 µg/ml in the absence of S9 and at 250 µg/ml in the presence of S9. Therefore, for the first experiment doses of 150, 200, 250, 300, 400 and 450 µg/ml without S9 and 80, 100, 112.5, 125, 137.5, 150 and 200 µg/ml with S9 were tested for viability and trifluorothymidine (TFT) resistance. For the second experiment the following doses were plated for viability and TFT resistance: 100, 150, 200, 250, 300, 350, 400 and 450 µg/ml without S9 and 25, 50, 75, 100, 125, 150, 175 and 200 µg/ml with S9. The highest doses 450 and 500 µg/ml without S9 and 250 and 300 µg/ml with S9 were excluded from plating due to excessive cytotoxicity.

Vehicle / solvent:

DMSO- the test substance has a solubility of 500mg/ml in DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: with metabolic activation

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: without metabolic activation

Details on test system and experimental conditions:

A 3-hour treatment incubation period was employed for all treatments in the presence and absence of S9 mix. The highest dose of 500 µg/ml without S9 was considered too toxic and was excluded from viability and (TFT) plating. For the second experiment the following doses were plated for viability and TFT resistance: 100, 150, 200, 250, 300, 350, 400 and 450 µg/ml without S9 and 25, 50, 75, 100, 125, 150, 175 and 200 µg/ml with S9. The highest doses of 475 and 500 µg/ml without S9 and 250 and 300 µg/ml with S9 were excluded from plating due to excessive toxicity.

Evaluation criteria:

The test article was considered to be mutagenic if all the following criteria were met:
the assay was valid according to the Acceptance criteria; the mutation frequency at one or more doses was significantly greater than that of the negative control (p<0.05); there was a significant dose-relationship as indicated by the linear trend analysis (p<0.05)

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was carried out according to UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment dose based on Dunnett's test for multiple comparisons. The data was checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test is one tailed, therefore a negative trend wasnot considered significant. These tests require the calculation of a heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

In the absence of metabolic activation, there was no significant increase in mutation frequency in either the first or second experiment up to toxic doses. In the presence of S9, statistically significant increases in mutation frequency were observed at the highest doses in both the first (137.5, 150 and 200 µg/ml) and second (150, 175 and 200 µg/ml) experiment. The relative total growth (RTG) at these doses were 52, 58 and 41% in the first experiment and 28, 18 and 10% in the second experiment, respectively. Large and small colonies were scored for the doses at which statistically significant increases in mutation frequency were observed. Increases in both small and large colony mutant frequencies were noted as well as a clear increase in the proportion of small colony mutants, indicating potential clastogenic activity.

Conclusions:

When tested up to toxic doses, tris(2-chloro-1methylethyl) phosphate induced mutation at the tk locus of L5178Y mouse lymphoma cells in two independent experiments in the presence of S9, but did not induce mutation in two independent experiemnts in the absence of S9. It is concluded that under the conditons employed in the study, Tris(2-chloro-1-methylethyl) phosphate was mutagenic in this test system in the presence of S9, but not in the absence of S9.

Executive summary:

When tested up to toxic doses, tris(2-chloro-1methylethyl) phosphate induced mutation at the tk locus of L5178Y mouse lymphoma cells in two independent experiments in the presence of S9, but did not induce mutation in two independent experiemnts in the absence of S9. It is concluded that under the conditons employed in the study, Tris(2-chloro-1-methylethyl) phosphate was mutagenic in this test system in the presence of S9, but not in the absence of S9.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Sponsor's ID: TMCPP
Chemical Name: Tris-(Beta-chloropropyl)phosphate
Purity: >99%
Date Received: 3 Aug 1992
Description: colourless liquid
Container: brown glass bottle
Storage conditions: room temperature

Target gene:

histidine (Salmonella typhimurium), tryptophan (E.coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9.

Test concentrations with justification for top dose:

8, 40, 200, 1000 and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

N-ethyl-N¿-nitro-N-nitrosoguanidine (TA 100, TA 1535 and WP2uvrA-), 9-aminoacridine (TA 1537) and 4-nitroquinoline-1-oxide (TA 98); 2-aminoacridine (TA 1535 and WP2uvrA-) and benzo[a]pyrene (TA 100, TA 1537 and TA 98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: ; in agar (plate incorporation);


DURATION
- Preincubation period: N/A
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent):N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A


SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A





DETERMINATION OF CYTOTOXICITY
- preliminary toxicity screen by plate incorporation, counting revertant colonies and background lawn assessment

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:

Evaluation criteria:

Positive result:A dose related and statistically significant increase in mutation rate ( at least two fold the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels.
Negative result: the number of induced revertants compared to the spontaneous revertants should be less than two-fold at each dose level employed, upto the dose limit (as determined by toxicity, solubility or limit dose of 5000µg/plate).

Statistics:

Statistics not performed

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

negative

Conclusions:

Interpretation of results (migrated information):
negative

The test material, TMCPP, was found to be non-mutagenic under the conditions of the test.

Executive summary:

The test material, TMCPP, was found to be non-mutagenic under the conditions of the test.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

OECD 489 (equiv.) (Covance Laboratories Ltd, 2006): Negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: other: DNA Damage and/or repair; Organ: liver.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed to Guideline.

Qualifier:

according to guideline

Guideline:

other: recommendations of IWGTP workshop

Principles of method if other than guideline:

The Comet assay is a technique for investigating DNA breakage and damage in individual mammalian cells by using electrophoresis of DNA which has been unwound under alkaline conditions (> pH 13). Electrophoresis results in the charged DNA being drawn away from the nucleus, with relaxed and broken DNA fragments migrating further than undamaged DNA complexes. The use of alkaline conditions enables single stranded and alkaline labile sites as well as double stranded DNA breaks to be expressed during electrophoresis.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

A sufficient number of out-bred 7-8 week old adult male Sprague Dawley Crl:CD® rats were obtained from Charles River UK Ltd, Margate, UK. They were housed in cages that conformed to the Code of Practice for the housing and care of animals used in scientific procedures (Home Office, London, 1989). They were housed in groups of six. Aspen wood chips (Datesand Ltd, Manchester) were used for bedding. Bottled water (public supply) and diet, (Special Diets Services Ltd, RM1.(E).SQC.) were provided ad libitum. Additionally, in order to enrich the environment and enhance the welfare of the animals, they were provided with wooden Aspen chew blocks and/or nesting material. All of the above were routinely monitored and are not known to contain any biological or chemical entity which might have interfered with the test system.
Routinely, the temperature and relative humidity was 19 to 25°C and 40 to 70%, respectively. No deviation from these ranges occurred for longer than 24 hours throughout this study. The holding rooms were illuminated by fluorescent light for 12 hours out of each 24 hour cycle and are designed to receive at least 15 fresh air changes per hour.
Animals were acclimatised for at least 5 days prior to dosing and were uniquely identified by numbered ear-tag. Rats were individually identified and randomised to groups of six animals using a system of random numbers. Cages were suitably labelled (using a colour-coded procedure) to clearly identify the study number, study type, start date, number and sex of animals, together with a description of the dose level and proposed time of necropsy. Checks were made on the first day of treatment to ensure group weights differed from the overall mean by no more than 5%.
Number of animals used in study
42Males
Weight range on first day of assay (g)
224-268
Approximate age on first day of assay (weeks)
7-8
Acclimatisation period (days)
8

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

groups of six male rats were administered TCPP in corn oil as a single dose via oral gavage at either 750 or 1500 mg/kg. The choice of dose levels was based on previous toxicity studies on TCPP, which identified 1500 mg/kg as the maximum tolerated dose.
Ethylmethanesulfonate was used as the positive control, dissolved at 25mg/ml in purified water.

Duration of treatment / exposure:

The animals were sacrificed in group order, 3or 24 hours after administration. Positiver control animals were killed 3 hours after administration.

Frequency of treatment:

Single dose

Post exposure period:

3 hours or 24 hours

Remarks:

Doses / Concentrations:
750 mg/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

6 males

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control group received a single gavage dose of ethyl methansulfonate (EMS) at 250 mg/kg three hours prior to necropsy.

Tissues and cell types examined:

The liver was chosen for comet analysis as TCPP caused an increased mutation frequency in the mouse lymphoma assay in the presence of S9 and induced liver enlargement in repeated dose toxicity studies.

Details of tissue and slide preparation:

The TCPP or the vehicle control treated rats were killed 3 or 24 hours after dosing. At necropsy, TCPP animals were examined internally for signs of cytotoxicity. For each animal, a section of the liver was removed, cut into small pieces and pushed through bolting cloth of pore size 150 µm to produce single cell suspensions. An aliquot of the cell suspension was then added to agarose, plated onto four slides and allowed to gel. Three slides were placed in lysis buffer for 1 hour, then transferred to electrophoresis buffer (pH > 13) to allow DNA to unwind for 30 minutes, after which the slides were electrophoresed at 0.7 V/cm for 40 minutes. At the end of the electrophoresis period slides were neutralised, dried and stained with ethidium bromide for comet analysis. The fourth slide was neutralised and used to determine the degree of highly damaged cells in the cell suspensions (diffusion analysis). Scoring of slides was made using fluorescence microscopy at x 200 magnification and Comet scoring was performed using Perceptive Instruments ¿Comet Assay III¿ image analysis system. Measurements of tail moment and tail intensity (% DNA in tail) were obtained from 100 cells per animal.

Evaluation criteria:

The tail moment is defined as [tail profile centre of gravity ¿ head profile centre of gravity] x tail % DNA, and therefore gives a measure incorporating both tail length and tail content. Each slide was also examined for possible indications of cytotoxicity, with cells with ¿clouds¿, which is a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis, were not included in Comet scoring. Diffusion slides were scored by assessing 100 cells per slide.

Statistics:

Treatment of data
The experimental unit of exposure for in vivo studies is the animal, and all analysis was based on individual animal response. The following was calculated for each animal.
tail moment
tail intensity (i.e. % DNA in the tail)
Values obtained from each parameter were treated as follows:
1. Values were added together from replicate slides
2. The median was calculated
3. The mean of the medians and standard error of the mean was calculated for each group.
No further statistical analysis was performed.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Lethargy was observed in one animal at 1500 mg/kg, with no other clinical signs noted. At necropsy, the livers of animals dosed at 1500 mg/kg were noted to be darker in appearance than those of the 750 mg/kg or vehicle control groups. Cloud assessment and analysis of diffusion slides of TCPP and vehicle control treated animals demonstrated low levels of cells (less than 15%) with significantly fragmented DNA, indicating little cytotoxicity, necrosis or apoptosis in the cell preparations. Comet analysis of livers treated with TCPP, sampled at either 3 or 24 hours post dosing, had slightly elevated group mean tail moments and intensities compared with the concurrent control. However, there was no dose response, the increases were within the degree of variation frequently seen with this assay and also fell within the historical control range. The positive control induced a clear increase in tail moment and tail intensity.Table 4.45below summarises the group mean data, including tail moment and tail intensity values.

Table 4.45  Summary of group mean data forin vivoComet assay with TCPP

Treatment group (mg/kg/day)	Sample time (hrs after dosing)	Group mean % clouds	Group mean % diffused cells	Tail Moment ± SEM	Tail Intensity ± SEM
Vehicle (0)	3	2.17	6.33	0.29 ± 0.04	2.20 ± 0.20
TCPP (750)	3	3.08	4.83	0.48 ± 0.04	2.94 ± 0.12
TCPP (1500)	3	2.50	8.83	0.51 ± 0.05	3.46 ± 0.25
Positive control	3	2.17	11.33	1.40 ± 0.05	6.77 ± 0.31
Vehicle (0)	24	2.17	5.50	0.41± 0.04	2.91 ± 0.20
TCPP (750)	24	1.42	6.67	0.41 ± 0.02	2.90 ± 0.14
TCPP (1500)	24	1.33	7.50	0.49 ± 0.05	3.29 ± 0.32

Conclusions:

Interpretation of results (migrated information): negative
It was concluded that TCPP did not induce DNA damage in the liver or rats treated up to 1500 mg/kg.

Executive summary:

The potential of TCPP to induce DNA strand breaks or alkali sites by the degree of DNA damage in the liver of treated rats was investigated in the Comet Assay.

The choice of dose levels was based on toxicity studies, which identified 1500 mg/kg as the maximum tolerated dose. In this study, groups of six male rats were administered TCPP at 750 and 1500 mg/kg. The test article was formulated in corn oiland administered as a single dose via oral gavage at a dose volume of 10 mL/kg. The liver of the treated rats was analysed for DNA damage 3 or 24 hours after dose administration. Lethargy was observed in one of the animals receiving the highest dose of TCPP.

The negative (vehicle) control in the study, corn oil, was also administered once via oral gavage to groups of six rats that were killed and sampled 3 or 24 hours after dose administration. Ethyl methanesulfonate (EMS), the positive control, was dissolved in purified water and administred via oral gavage as a single dose at 250 mg/kg (dose volume of 10 mL/kg) to a group of six rats which were killed 3 hours after dose administration.

It is concluded that , unted the conditions of this Comet Assay, TCPP did not induce DNA damage in the liver of rats treated up to 1500 mg/kg/day, when analysed 2 hours or 24 hours after dose administration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not applicable

Additional information

Hazards identified by EU Risk Assessment in May 2008:

"The mutagenic potential of TCPP has been well investigated in vitro. Evidence from several bacterial mutagenicity studies shows that TCPP is not a bacterial cell mutagen. TCPP was also shown to be non-mutagenic in fungi. In mammalian cell studies, TCPP did not induce forward mutations at the TK locus in L5178Y mouse lymphoma cells in one study, but in a second study, the result was considered equivocal (in the presence of rat liver S9 fraction). A confirmatory mouse lymphoma was conducted in accordance with the relevant regulatory guidelines. The results of the assay indicate that TCPP shows clastogenic activity in vitro in the presence of metabolic activation. In one GLP study, TCPP did not induce unscheduled DNA synthesis in vitro. Two other in vitro UDS studies are reported. In one, TCPP gave a negative result; in the second, the result is considered equivocal. In an in vitrotransformation assay, TCPP was seen to induce transformed foci in BALB/3T3 cells, whereas in another similar study, it did not. As indicated above, the results of the most recent in vitro mouse lymphoma assay were positive. In particular in this study, there was a clear increase in the proportion of small colony mutants, giving rise to concern for a possible clastogenic effect of TCPP. Due to this positive study, industry proceeded to carry out the above-mentioned in vitro/in vivo UDS assay to further investigate the mutagenic potential of TCPP in vivo. In this study, statistically significant increases in NNG and a dose response effect at one time point were observed. However, as the counts did not exceed zero at either of the doses tested, the biological significance of the effect is doubtful and thus the result is considered equivocal. The main concern for TCPP is clastogenicity, owing to the clearly positive in vitro mouse lymphoma study. The UDS assay is not considered to be the most appropriate test for investigating a potential clastogen, as clastogenic substances are not expected to be efficient at inducing unscheduled DNA synthesis. In vivo, TCPP was not clastogenic in a mouse bone marrow micronucleus test. TCPP did not induce an increase in chromosomal aberrations in a rat bone marrow cytogenetics assay. However, there were some shortcomings in these studies and it is considered that they do not fulfil all current regulatory guidelines as described in the study summaries in 4.1.2.7.2. Therefore, in order to investigate the potential for TCPP to induce DNA damage, an in vivo Comet assay in the rat liver was conducted. The liver was chosen for comet analysis as TCPP caused an increased mutation frequency in the mouse lymphoma assay in the presence of S9 and also induced liver enlargement in repeat dose studies. Under the conditions of this study, TCPP did not induce DNA damage in the liver of rats treated with either 750 or 1500 mg/kg TCPP. Overall, it is considered that TCPP is not genotoxic in vivo."

For detailed information of studies mentioned in the Summary of the EU RAR (see above) but not in Table 5.7.1.1 or 5.7.1.2. of the CSR see Iuclid chapter 7.6.1 and 7.6.2.

 

Updated relevant information (March 2018):

Tris(chloropropyl)phosphate (M20263) [= TCPP] is on the Testing List of the National Toxicology Program (NTP) with, amongst others, peripheral blood micronucleus tests in rats and mice. According to NTP these studies are completed, but up to now only visible in table form, see description below.

http://ntp.niehs.nih.gov/testing/status/agents/ts-m20263.html

Preliminary results obtained in a peripheral blood micronucleus tests on rats were reported on the NTP-webpage. The study was included in a rat NTP 13-week toxicity study as part of the bioassay program. At the end of the 13-week exposure period (route of exposure: dosed-feed) a blood sample was obtained from male and female rats in each dose group (5 animals per treatment group per sex). 1000 to 10000 mature erythrocytes (normochromatic erythrocytes or NCEs) were scored per animal for presence of micronuclei. Doses of 1250 up to 10000 ppm for male rats and 20000 ppm for female rats were tested. There is no information available yet on the actual ingested doses.

The test result was negative for male and female rats and thus TCPP was shown to be not mutagenic in the peripheral blood micronucleus test in rats.

Preliminary results obtained in a peripheral blood micronucleus tests on mice were also reported on the NTP-webpage. The study was included in a mouse NTP 13-week toxicity study as part of the bioassay program. At the end of the 13-week exposure period (route of exposure: dosed-feed), a blood sample was obtained from male and female mice in each dose group (5 animals per treatment group per sex). 1000 to 10000 mature erythrocytes (normochromatic erythrocytes or NCEs) were scored per animal for presence of micronuclei. Doses of 1250 up to 20000 ppm were tested in both sexes. This corresponds to actual ingested doses of about 500, 1000, 2400 and 4500 mg/kg bw/day for males and 450, 900, 1800, and 3500 mg/kg bw/day for females according to a rough calculation from the tables shown.

The test result was clearly negative for female mice. In male mice the mean frequency of micronucleated polychromatic erythrocytes (MN PCEs)/1000 PCEs was 3.2 in the high-dosed animals compared to 2.6 in the control group. The high-dosed animals showed a range of MN PCEs of 2.5 to 3.7 with a statistical significance of p<0.05. The range of MN PCEs in the control animals was 2.2 to 3.2, i.e. that the ‘positive’ mean of 3.2 is within the control range and thus is only a very slight effect with questionable biological relevance. In the clearly ‘negative’ dose-group of 5000 ppm the MN-PCE range was 1.8 to 3.8 (mean 2.5). Additionally the high-dosed animals showed distinct signs of toxicity at this dose (body weight 25% lower than control males; liver and kidney toxicity). The slightly increased frequency of MN PCEs in male mice only is thus considered to be of minor relevance. For a final assessment the historical control data of the laboratory are requested. An FOIA request was send to NTP in February 2018 to get the historical control data for rat and mice peripheral blood micronucleus assays.

Justification for classification or non-classification

EU Risk Assessment of 2008: Based on the available studies, TCPP needs no classification for mutagenicity according to EU guidelines.

Update March 2018: The conclusion of non-classification for mutagenicity is confirmed and compliant with EU Regulation 1272/2008.