4-chloroformylphthalic anhydride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 27 June to 18 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Remarks:

/ in vivo Micronucleus test combined with in vivo Comet assay

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 27 June to 18 August 2022

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to same study

Remarks:

/ in vivo Micronucleus test combined with in vivo Comet assay

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

This comet assay was combined with micronucleus test (according to the guideline OECD 474).

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo, Blackthorn, UK.
- Age at study initiation: 6 to 8 weeks old for the range-finder experiment and 7 to 8 weeks old for the main experiment
- Weight at study initiation: 182-197 g (males) or 162-173 g (females) for the range-finder experiment and 215-256 g (males) for the main experiment
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: in wire topped, solid bottomed cages, with three animals of the same sex per cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Animals were acclimatised for at least 5 days and a health inspection was performed before the start of dosing to ensure their suitability for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light

IN-LIFE DATES:
Study Initiation Date: 20 June 2022
Experimental Start Date: 27 June 2022
In-life Start Date: 27 June 2022
In-life End Date: 14 July 2022
Experimental Completion Date: 18 August 2022

Route of administration:

oral: gavage

Vehicle:

- Vehicle: corn oil
- Justification for choice of solvent/vehicle: common organic solvent used for oral dosing in rodents and has been used in previous in vivo toxicity studies with compounds that were close analogues of the test article.
- Concentration of test material in vehicle: 35, 70 and 140 mg/mL
- Dose volume: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was weighed out into a pre-labelled container and vehicle added to achieve the final volume. The mixture was stirred vigorously and heated to approximately 75°C (77°C, 78°C or 88°C for Main Experiment formulations) until visibly homogenous, then allowed to cool to room temperature whilst stirring. Formulations were then aliquoted as required.

STABILITY OF DOSING FORMULATIONS
Prior to the Main Experiment, Trimellitic anhydride chloride (TMAC) formulations in the vehicle at 10, 100 and 200 mg/mL were confirmed as stable and homogenous for 24 hours when stored at 15 to 25⁰C, and for 4 days when stored refrigerated (2 to 8°C) and protected from light.
All formulations used for animal dosing in the Main Experiment were protected from light and stored 2-8°C when not required for dosing. On each day of dosing of the Main Experiment, an aliquot of each formulation was held at room temperature in the animal facility and used on the day of removal from refrigerated conditions.
All formulations were used within 3 days of preparation.

Duration of treatment / exposure:

once daily administration, for 3 days (at 0, 24 and 45h)

Frequency of treatment:

daily

Post exposure period:

not applicable

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control group

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose) = 266 mg/kg bw/day

Dose / conc.:

700 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose) = 700 mg/kg bw/day

Dose / conc.:

1 400 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose) = 1400 mg/kg bw/day

No. of animals per sex per dose:

6 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

ethyl methanesulfonate
- Route of administration: oral (gavage)
- Doses / concentrations: 150 mg/kg bw/day
- Prepared in water at the concentration of 15 mg/mL

Tissues and cell types examined:

Liver, glandular stomach and duodenum
Germs cells were collected but not analyzed as results in somatic cells are negative.

Details of tissue and slide preparation:

PREPARATION OF COMET SLIDES SUSPENSIONS
Tissues were placed in pots appropriately covered in Merchants + DMSO (hereafter known as ‘Merchants’) solution in necropsy and held on ice until processed by Genetic Toxicology.

> The comet liver samples were cut into small pieces and washed thoroughly in Merchants solution. The pieces of liver were minced into smaller pieces and then pushed through bolting cloth (pore size of 150 µm) to produce single cell suspensions.
> The comet glandular stomach samples were incubated on ice for 15 minutes prior to processing. After incubation the stomach samples were removed from the Merchants solution and the inner surface gently scraped twice using the back of a scalpel blade. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade in 200 μL of fresh Merchants solution to produce single cell suspensions.
> The comet duodenum samples were vortexed in ice cold Merchants solution for approximately 20 seconds. The tissue was removed from the Merchants solution and the inner surface gently scraped using the back of a scalpel blade. The tissue was vortexed in ice cold Merchants solution for a further 20 seconds prior to gently scraping the inside of the duodenum with the back of a scalpel blade in 150 μL of fresh Merchants solution to produce single cell suspensions.
> The comet gonad samples were prepared by making an incision along the length of a single gonad, removing the contents from the membrane and discarding the membrane. The remaining tissue was cut into small pieces and gently pushed through bolting cloth (pore size of 150 μm) with approximately 10 mL of Merchants solution to produce single cell suspensions.

All cell suspensions were held on ice prior to slide preparation.

PREPARATION OF COMET SLIDES
Three slides were prepared per single cell suspension per tissue. Slides were dipped in molten normal melting point agarose such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 µL of each single cell suspension was added to 400 µL of 0.7% low melting point agarose at approximately 37°C. 100 µL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.

CELL LYSIS
Once gelled the coverslips were removed and all slides placed in lysis buffer overnight at 2-8°C, protected from light.

UNWINDING AND ELECTROPHORESIS
Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer at 2-8°C and the DNA unwound for 20 minutes (glandular stomach and duodenum) or 30 minutes (liver and gonad). At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes (glandular stomach and duodenum) or 40 minutes (liver and gonad). As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

NEUTRALISATION
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0. After neutralisation the slides were dried and stored at room temperature prior to scoring.

STAINING
Prior to scoring, the slides were stained with 100 µL of 2 µg/mL ethidium bromide and coverslipped.

SLIDES ANALYSIS
Scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters for the stains used.

SCORING OF COMETS
Measurements of tail intensity (%DNA in tail) were obtained from 150 cells/animal. In general, this was evenly split over three slides.
The number of ‘hedgehogs’ observed during comet scoring was recorded for each slide. To avoid the risk of false positive results ‘hedgehogs’ were not used for comet analysis. Each slide was scanned starting to the left of the centre of the slide.
The following criteria were used for analysis of slides:
1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored.

Evaluation criteria:

ACCEPTANCE CRITERIA (STUDY VALIDITY)
The data were considered valid if the following criteria were met:
1. The vehicle control data were comparable to the laboratory historical control data for each tissue
2. The positive control induced responses that were compatible with the laboratory historical control data and that produced a statistically significant increase compared to the concurrent vehicle control
3. Adequate numbers of cells and doses were analyzed
4. The high dose was considered to be the MTD, the maximum recommended dose, the maximum practicable dose or one that demonstrated cytotoxicity to the target cells.

ACCEPTANCE CRIETRIA FOR DNA DAMAGE
For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose related in any tissue
3. The increase exceeded the laboratory’s historical control data for that tissue.
The test article was considered positive in this study if all the criteria listed were met.
The test article was considered negative in this study if for both end points, none of the following criteria were met and target tissue exposure was confirmed.

Statistics:

After completion of microscopic analysis and decoding of the data the percentage tail intensity (i.e. %DNA in the tail) was calculated.
Data were treated as follows:
1. The median value per slide was calculated
2. The mean of the slide medians was calculated to give the mean animal value
3. The mean of the animal means and standard error of the mean was calculated for each group.
Tail intensity data for each slide were supplied for statistical analysis. The median of the log-transformed tail intensities from each slide was averaged to give an animal summary statistic. Where the median value on a slide was zero, a small constant (0.0001) was added before taking the logarithm and calculating the average for the animal. This animal average was used in the statistical analysis.
Data was analysed using one-way analysis of variance (ANOVA) with the fixed factor for treatment group. The positive control group was excluded from this analysis. Levene’s test was used to test for equality of variances among groups. This showed no evidence of heterogeneity (P>0.01). Comparisons between each treated group and control were made using Dunnett’s test. The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p value are reported. In addition, a linear contrast was used to test for an increasing dose response.
The positive control group was compared to the control group using a two-sample t test. Levene’s test was used to test for equality of variances between the groups. This showed no evidence of heterogeneity (P>0.01). The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p-value are reported.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

GLANDULAR STOMACH

Toxicity:

yes

Remarks:

Microscopic findings in the stomach were reported (erosion, inflammation and vesicle formation in the forestomach / minimal to moderate degeneration/atrophy in the glandular stomach).

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

DUODENUM

Toxicity:

no effects

Remarks:

no macroscopic or microscopic effects were reported on the duodenum.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

LIVER

Toxicity:

yes

Remarks:

Decrease of glycogen and variations in liver enzymes activity were reported.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
See above in the section "Any other information on materials and methods incl. tables".
A preliminary experiment was conducted at 2000 and 1400 mg/kg bw/day.
On Day 3, there were significant observations in both sexes for animals administered 2000 mg/kg (mouth rubbing, ataxia, decreased activity, excessive salivation, diarrhoea, raised hair, semi-closed eyes, red/brown mouth and/or snout, audible breathing, paddling, vocalisation, hunched posture and/or prone; two females were found dead 2 hours post dose). Therefore, 2000 mg/kg/day clearly exceeded an appropriate maximum tolerated dose in both sexes. As such, bone marrow toxicity was not assessed in surviving animals.
From these results, 1400 mg/kg/day was considered to be an appropriate estimate of the MTD and was therefore selected as the maximum dose for the Main Experiment. Two lower doses of 350 (25% MTD) and 700 mg/kg/day (50% MTD) were selected.


RESULTS OF THE MAIN EXPERIMENT
- POST DOSE OBSERVATIONS
There were no clinical observations of toxicity on Day 1 or Day 2 for any animal dosed in the Main Experiment. On Day 3, clinical observations at 1400 mg/kg/day included liquid or soft faeces, mouth rubbing, mild decreased activity, raised hair, ataxia and low carriage. Transient observation of mouth rubbing was noted for animal R0206 at 700 mg/kg/day on Day 3.

- BODY WEIGHTS
There was a test article-related effect on animal body weight between Day 1 – Day 3 with group mean body weight change values of +5.0%, +4.4% and -1.1% at 266, 700 and 1400 mg/kg/day, respectively, compared to +4.2% in the concurrent vehicle control group.

- CLINICAL CHEMISTRY
Increased alanine aminotransferase (ALT) activity and decreased alkaline phosphatase (ALP) activity were observed for animals administered 700 or 1400 mg/kg/day. Increased cholesterol was recorded for animals administered 1400 mg/kg/day.
Decreased albumin and increased globulins were noted for animals administered 1400 mg/kg/day, with a decreased albumin:globulin ratio.
Decreased sodium and increased potassium were noted for animals administered 1400 mg/kg/day. Increased chloride was noted for animals administered 700 or 1400 mg/kg/day.
Increased calcium and phosphate were noted for two animals administered 1400 mg/kg/day.
Increased creatinine was noted for animals administered 1400 mg/kg/day. Increased urea was noted for animals administered 700 or 1400 mg/kg/day.
Increased glucose was noted for animals in all groups administered TMAC, with a dose relationship. Glucose was particularly high for Animal R0306 (34.7 mmol/L). This was considered indicative of a stress response.

- HISTOPATHOLOGY
> On macroscopic examination, cysts were noted in the cardia of the stomach of two animals administered 700 mg/kg/day, which correlated with microscopic vesicles. Red foci in the stomach of one animal administered 1400 mg/kg/day correlated with microscopic erosions/ulcers.
> On microscopic examination, TMAC-related changes were recorded for the liver and stomach.
Decreased hepatocyte glycogen was recorded for animals from all groups administered Trimellitic anhydride chloride (TMAC), with a dose relationship.
In the stomach, erosion/ulcer, inflammation, and vesicle formation of the forestomach were noted in animals from all groups administered Trimellitic anhydride chloride (TMAC) generally with a dose relationship. More vesicles were noted in animals administered 700 mg/kg/day, as ulceration in the high-dose group effaced the epithelial surface of the forestomach. In the glandular stomach, minimal to moderate degeneration/atrophy was noted in all groups administered TMAC. Occasional erosions/ulcers were noted in animals administered 700 or
1400 mg/kg/day.
> In absence of any genotoxic effect in somatic cells, germ cells were not analyzed.

- CONCLUSION ON TOXICITY
Findings in the stomach are consistent with an irritant or even corrosive effect of the test article.
In conclusion, TMAC was associated with increased ALT and decreased ALP activities; increased cholesterol, globulins, potassium, chloride, calcium, phosphate, urea, and creatinine; decreased albumin and sodium; macroscopic cysts in the stomach; erosions/ulcers, inflammation, vesicles, and degeneration atrophy in the stomach; and decreased glycogen in the liver.

- CONCLUSION ON VALIDITY OF THE STUDY AND EXPOSURE OF TARGET TISSUES
The data generated in this study confirm that:
1. The vehicle control data were comparable to laboratory historical control data for each tissue
2. The positive control induced responses that were compatible with the laboratory historical control data and are statistically significant compared to the concurrent vehicle control
3. Adequate numbers of cells and doses were analysed
4. The high dose was considered to be the MTD.

As dosing was via oral gavage, exposure to the glandular stomach and duodenum was assured. Furthermore, microscopic findings were observed in the stomach in response to test article exposure. With regards to the liver, decreased hepatocyte glycogen indicated liver perturbations following dosing, which was attributed directly or indirectly to the effects of the test article. Furthermore, liver enzyme (ALT and ALP) activity was impacted at the
intermediate and high dose group providing evidence of a toxicological response. The clinical chemistry, histopathology, clinical observations and body weight effects combined provide indirect and direct evidence of systemic test article exposure.
The assay data were therefore considered valid.

- GENETIC TOXICITY - COMET ANALYSIS
There were no marked increases in %hedgehogs in the liver, glandular stomach or duodenum that were considered indicative that treatment with Trimellitic anhydride chloride (TMAC) induced excessive DNA damage that could have interfered with comet analysis.
Animals treated with Trimellitic anhydride chloride (TMAC) at 266, 700 or 1400 mg/kg/day exhibited group mean percentage tail intensities in the liver, glandular stomach and duodenum that were similar to the concurrent vehicle control group mean and that fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response.
It was noted that there were elevated individual animal values in the liver above the 95% reference range in the vehicle control group and each test article dosed group. As these values were observed in each group and not replicated across the majority of animals in each group, they were considered not related to Trimellitic anhydride chloride (TMAC) treatment and therefore of no biological relevance.
In absence of any genotoxic effect in somatic cells, germ cells were not analyzed.

- FORMULATIONS ANALYSIS
Test article formulations at 35, 70 and 140 mg/mL were prepared once for dosing in the Main Experiment. The analytical data confirmed that dosing formulations at 70 and 140 mg/mL were homogenous (0.83-1.29% RSD; target ≤5% RSD) and achieved target nominal concentration, mean values of 88-91% (within 100±15%). However, while the formulation at 35 mg/mL was also homogenous (1.43% RSD), it fell outside acceptance criteria of 85-115% with a mean value of 76% and therefore an actual dose level of 266 mg/kg/day.

As there were no analytical errors found upon investigation, the analytical data were considered accurate for reporting. Although the dose level of 350 mg/kg/day was not achieved, there is no impact to the validity of the study as the formulation was homogenous such that there were no concerns over the actual dose provided (266 mg/kg/day), which is within acceptable dose level intervals per OECD guidelines (i.e. within 4-fold of 700 mg/kg/day).

The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

TMAC - Summary of Group Mean Comet Data – Liver

Group	Mean (SEM)	Back-Transformed Difference from Vehicle	Ranked	P-Value	Significance	Mean % Hedgehogs
1/ Vehicle 0 mg/kg bw/day	1.45 (0.39)	-	-	-	-	1.43
2/ TMAC 266 mg/kg bw/day	1.37 (0.54)	0.74	U	0.9209	NS	1.36
3/ TMAC 700 mg/kg bw/day	1.43 (0.57)	0.77	U	0.9096	NS	0.98
4/ TMAC 1400 mg/kg bw/day	1.74 (0.43)	1.08	U	0.6834	NS	2.38
5/ Positive (EMS) 150 mg/kg bw/day	46.81 (1.96)	39.18	U	< 0.0001	***	3.50
Dose response (groups 1, 2, 3, 4)			U	0.4270	NS	NA

 

TMAC - Summary of Group Mean Comet Data – Glandular stomach

Group	Mean (SEM)	Back-Transformed Difference from Vehicle	Ranked	P-Value	Significance	Mean % Hedgehogs
1/ Vehicle 0 mg/kg bw/day	0.85 (0.23)	-	-	-	-	7.24
2/ TMAC 266 mg/kg bw/day	0.90 (0.41)	0.77	U	0.8901	NS	10.94
3/ TMAC 700 mg/kg bw/day	0.78 (0.19)	0.87	U	0.8330	NS	8.45
4/ TMAC 1400 mg/kg bw/day	0.87 (0.33)	0.85	U	0.8466	NS	10.62
5/ Positive (EMS) 150 mg/kg bw/day	14.26 (2.22)	21.83	U	< 0.0001	***	10.62
Dose response (groups 1, 2, 3, 4)			U	0.5854	NS	NA

 

TMAC - Summary of Group Mean Comet Data – Duodenum

Group	Mean (SEM)	Back-Transformed Difference from Vehicle	Ranked	P-Value	Significance	Mean % Hedgehogs
1/ Vehicle 0 mg/kg bw/day	0.88 (0.20)	-	-	-	-	4.48
2/ TMAC 266 mg/kg bw/day	0.36 (0.07)	0.36	U	0.9983	NS	3.12
3/ TMAC 700 mg/kg bw/day	0.56 (0.11)	0.60	U	0.9686	NS	2.65
4/ TMAC 1400 mg/kg bw/day	0.77 (0.49)	0.40	U	0.9964	NS	5.54
5/ Positive (EMS) 150 mg/kg bw/day	12.99 (1.34)	18.03	U	< 0.0001	***	8.98
Dose response (groups 1, 2, 3, 4)			U	0.9190	NS	NA

NA: not applicable / SEM: Standard Error of Mean / EMS: Ethyl Methanesulfonate

NS: Not significant (P>0.05) / U: Unranked / ***: P <= 0.001

 

TMAC - Summary of Body Weight Percentage Change

Group	N	Mean % change (SD)
1/ Vehicle 0 mg/kg bw/day	6	4.2 (1.36)
2/ TMAC 266 mg/kg bw/day	6	5.00 (1.11)
3/ TMAC 700 mg/kg bw/day	6	4.4 (1.89)
4/ TMAC 1400 mg/kg bw/day	6	-1.1 (3.56)
5/ Positive (EMS) 150 mg/kg bw/day	6	-7.4 (2.31)

SD: standard deviation

N: number of individual values (animals)

Conclusions:

When tested in the Comet assay conducted according to the OECD 489 guideline in rats exposed up to 1400 mg/kg bw/day (maximum tolerated dose), Trimellitic anhydride chloride (TMAC), did not induce DNA strand breaks in the liver, glandular stomach or duodenum. In absence of any effect in somatic cells, germ cells were not analyzed.

Executive summary:

Trimellitic anhydride chloride (TMAC) was tested for its potential to induce DNA strand breaks in the liver, stomach and duodenum of rats. The study was conducted according to the guideline OECD 489 and combined with the micronucleus test (according to the guidelien OECD 474).

Male Sprague Dawley rats were treated by gavage once daily, for 3 days, at the actual dose levels of 266, 700 and 1400 mg/kg bw/day. Based on a range-finder experiment, the dose level of 1400 mg/kg bw/day was considered as being the MTD (maximum tolerated dose). The vehicle control group received the vehicle (corn oil) and the positive control group received Ethyl methanesulfonate (EMS) at 150 mg/kg bw/day.

Tissues (liver, glandular stomach, duodenum and germ cells) were collected at necropsy (Day 3, 48 hours, i.e. 3 hours after the last dosing).

There were no clinical observations of toxicity on Day 1 or Day 2 for any dosed animal. On Day 3, clinical observations at 1400 mg/kg/day included liquid or soft
faeces, mouth rubbing, mild decreased activity, raised hair, ataxia and low carriage. 

There was a test article-related effect on animal body weight between Day 1 – Day 3 with group mean body weight change values of +5.0%, +4.4% and -1.1% at 266, 700 and 1400 mg/kg/day, respectively, compared to +4.2% in the concurrent vehicle control group.

Liver enzyme (ALT and ALP) activity was impacted at the intermediate and high dose group providing evidence of a toxicological response. Variations were also observed for cholesterol, albumin, sodium and potassium, calcium, phosphate and glucose mainly for animals administered 1400 mg/kg bw/day.

On microscopic examination, TMAC-related changes were recorded for the liver and stomach. Decreased hepatocyte glycogen was recorded for animals from all groups administered TMAC, with a dose relationship. In the stomach, erosion/ulcer, inflammation, and vesicle formation of the forestomach were reported generally with a dose relationship. In the glandular stomach, minimal to moderate degeneration/atrophy was noted in all groups administered TMAC.

All these findings combined (clinical chemistry, histopathology, clinical observations and body weight effects) provide indirect and direct evidence of systemic test article exposure.

There were no marked increases in %hedgehogs in the liver, glandular stomach or duodenum that were considered indicative that treatment with TMAC induced excessive DNA damage that could have interfered with comet analysis.

Animals treated with TMAC at 266, 700 or 1400 mg/kg/day exhibited group mean percentage tail intensities in the liver, glandular stomach and duodenum that were similar to the concurrent vehicle control group mean and that fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response. As no genotoxic effects were observed in somatic cells, the analysis of germ cells was not conducted.

It is concluded that under the conditions of this study, Trimellitic anhydride chloride (TMAC), did not induce DNA strand breaks in the liver, glandular stomach or duodenum when tested up to 1400 mg/kg/day (an estimate of the maximum tolerated dose for this study).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

This micronucleus test was combined with the comet assay (according to the guideline OECD 489).

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-chloroformylphthalic anhydride

EC Number:

214-874-8

EC Name:

4-chloroformylphthalic anhydride

Cas Number:

1204-28-0

Molecular formula:

C9H3ClO4

IUPAC Name:

1,3-dioxo-1,3-dihydro-2-benzofuran-5-carbonyl chloride

Details on test material:

trimellitic anhydride mono-chloride (TMAC)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo, Blackthorn, UK.
- Age at study initiation: 6 to 8 weeks old for the range-finder experiment and 7 to 8 weeks old for the main experiment
- Weight at study initiation: 182-197 g (males) or 162-173 g (females) for the range-finder experiment and 215-256 g (males) for the main experiment
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: in wire topped, solid bottomed cages, with three animals of the same sex per cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Animals were acclimatised for at least 5 days and a health inspection was performed before the start of dosing to ensure their suitability for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light

IN-LIFE DATES:
Study Initiation Date: 20 June 2022
Experimental Start Date: 27 June 2022
In-life Start Date: 27 June 2022
In-life End Date: 14 July 2022
Experimental Completion Date: 18 August 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: corn oil
- Justification for choice of solvent/vehicle: common organic solvent used for oral dosing in rodents and has been used in previous in vivo toxicity studies with compounds that were close analogues of the test article.
- Concentration of test material in vehicle: 35, 70 and 140 mg/mL
- Dose volume: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was weighed out into a pre-labelled container and vehicle added to achieve the final volume. The mixture was stirred vigorously and heated to approximately 75°C (77°C, 78°C or 88°C for Main Experiment formulations) until visibly homogenous, then allowed to cool to room temperature whilst stirring. Formulations were then aliquoted as required.

STABILITY OF DOSING FORMULATIONS
Prior to the Main Experiment, Trimellitic anhydride chloride (TMAC) formulations in the vehicle at 10, 100 and 200 mg/mL were confirmed as stable and homogenous for 24 hours when stored at 15 to 25⁰C, and for 4 days when stored refrigerated (2 to 8°C) and protected from light.
All formulations used for animal dosing in the Main Experiment were protected from light and stored 2-8°C when not required for dosing. On each day of dosing of the Main Experiment, an aliquot of each formulation was held at room temperature in the animal facility and used on the day of removal from refrigerated conditions.
All formulations were used within 3 days of preparation.

Duration of treatment / exposure:

once daily administration, for 3 days (at 0, 24 and 45h)

Frequency of treatment:

daily

Post exposure period:

Not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control group

Doses / concentrations 2

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose): 266 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

700 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose) = 700 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 400 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose) = 1400 mg/kg bw/day

No. of animals per sex per dose:

6 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

ethyl methanesulfonate
- Route of administration: oral (gavage)
- Doses / concentrations: 150 mg/kg bw/day
- Prepared in water at the concentration of 15 mg/mL

Examinations

Tissues and cell types examined:

Bone marrow cells were isolated from one femur

Details of tissue and slide preparation:

SAMPLING:
The isolated femur was cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrow was flushed from the marrow cavity with 2 mL fetal bovine serum into appropriately labelled centrifuge tubes. The samples were filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose. Once the majority of the 2 mL had passed through the column a further 4 mL of serum was added to the sample tubes and loaded onto the columns.
Once filtered, the bone marrow cells were pelleted by centrifugation (200 g, 5 minutes, 15-25°C) and the supernatant aspirated and discarded. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. The cells were pelleted again (as described above) and the supernatant aspirated to leave one or two drops and the cell pellet.

DETAILS OF SLIDE PREPARATION:
The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of two (Range-Finder Experiment) or three (Main Experiment) uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide.
Slides were air-dried, then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water. Two slides per animal were immediately stained for 5 minutes in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis.

METHOD OF ANALYSIS:
The relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 500 cells (PCE plus NCE) had been analysed as an indication of potential bone marrow toxicity. For the Main Experiment. analysis continued until at least 4000 PCE/animal were examined for the presence of MN. All PCE containing MN observed during these two phases of counting were recorded.

The following criteria were used for analysis of slides:
1. Cells were of normal cell morphology
2. Areas where erythrocytes overlapped were ignored
3. A MN was round or oval in shape
4. A cell containing more than one MN was scored as a single micronucleated cell
5. MN that were refractive, improperly stained or not in the focal plane of the cell were judged to be artefacts and were not scored.

Evaluation criteria:

ACCEPTANCE CRITERIA (STUDY VALIDITY)
The data were considered valid if the following criteria were met:
1. The vehicle control data were comparable to the laboratory historical control data for each tissue
2. The positive control induced responses that were compatible with the laboratory historical control data and that produced a statistically significant increase compared to the concurrent vehicle control
3. Adequate numbers of cells and doses were analysed
4. The high dose was considered to be the MTD, the maximum recommended dose, the maximum practicable dose or one that demonstrated cytotoxicity to the target cells.

CRITERIA FOR DNA DAMAGE
For valid data, the test article was considered to induce clastogenic/aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE exceeded the laboratory’s historical vehicle control data
3. A dose-response trend in the proportion of MN PCE (where more than two dose levels are analysed) was observed.

Statistics:

After completion of microscopic analysis and decoding of the data the following were calculated:
1. %PCE for each animal and the mean for each group. The group mean %PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity
2. Frequency of MN PCE (i.e. MN per number of PCE scored) and %MN PCE for each animal and the group mean %MN PCE (standard deviation).
The numbers of MN PCE in the vehicle control animals were compared with the laboratory's historical control data to determine whether the assay was acceptable. The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by use of the Wilcoxon rank sum test (Lehmann, 1975). The tests were interpreted with one-sided risk for increased frequency with increasing dose. A Terpstra-Jonckheere test was conducted to evaluate dose response (Jonckheere, 1954). Probability values of p≤0.05 were accepted as significant.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

A reduction of 13% was observed in group mean %PCE at 1400 mg/kg/day.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
See above in the section "Any other information on materials and methods incl. tables".
A preliminary experiment was conducted at 2000 and 1400 mg/kg bw/day.
On Day 3, there were significant observations in both sexes for animals administered 2000 mg/kg (mouth rubbing, ataxia, decreased activity, excessive salivation, diarrhoea, raised hair, semi-closed eyes, red/brown mouth and/or snout, audible breathing, paddling, vocalisation, hunched posture and/or prone; two females were found dead 2 hours post dose). Therefore, 2000 mg/kg/day clearly exceeded an appropriate maximum tolerated dose in both sexes. As such, bone marrow toxicity was not assessed in surviving animals.
From these results, 1400 mg/kg/day was considered to be an appropriate estimate of the MTD and was therefore selected as the maximum dose for the Main Experiment. Two lower doses of 350 (25% MTD) and 700 mg/kg/day (50% MTD) were selected.

RESULTS OF MAIN STUDY
> Post dose observation
There were no clinical observations of toxicity on Day 1 or Day 2 for any animal dosed in the Main Experiment. On Day 3, clinical observations at 1400 mg/kg/day included liquid or soft faeces, mouth rubbing, mild decreased activity, raised hair, ataxia and low carriage. Transient observation of mouth rubbing was noted for one animal at 700 mg/kg/day on Day 3.

> Toxicity - Ratio PCE/NCE
Animals treated with Trimellitic anhydride chloride (TMAC) at 266 or 700 mg/kg/day exhibited group mean %PCE that were similar to the vehicle control group mean and which were comparable with the laboratory’s historical vehicle.
A reduction of 13% was observed in group mean %PCE at 1400 mg/kg/day. This reduction was slight and within the laboratory’s historical vehicle control data. However, as it was observed in 5 out of 6 animals, it was considered potential evidence of bone marrow toxicity.

> Genotoxicity - Induction of micronuclei
Animals treated with Trimellitic anhydride chloride (TMAC) at 266, 700 or 1400 mg/kg/day exhibited group mean %MN PCE frequencies that were similar to the vehicle control group mean and which fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control with no evidence of a dose response. Individual animal MN PCE frequencies were considered consistent with the laboratory's historical vehicle control animal distribution data. It was noted that there was an isolated animal with a %MN PCE value (0.50%) that exceeded the 95% reference range (0-0.33%); however, as this elevated value was not reproduced in any other animal in the high dose group and the group data was not statistically increased compared to the concurrent vehicle control group, this value was considered of no biological relevance.

> Results of formulations analysis
Test article formulations at 35, 70 and 140 mg/mL were prepared once for dosing in the Main Experiment. The analytical data confirmed that dosing formulations at 70 and 140 mg/mL were homogenous (0.83-1.29% RSD; target ≤5% RSD) and achieved target nominal concentration, mean values of 88-91% (within 100±15%). However, while the formulation at 35 mg/mL was also homogenous (1.43% RSD), it fell outside acceptance criteria of 85-115% with a mean value of 76% and therefore an actual dose level of 266 mg/kg/day.
As there were no analytical errors found upon investigation, the analytical data were considered accurate for reporting. Although the dose level of 350 mg/kg/day was not achieved, there is no impact to the validity of the study as the formulation was homogenous such that there were no concerns over the actual dose provided (266 mg/kg/day), which is within acceptable dose level intervals per OECD guidelines (i.e. within 4-fold of 700 mg/kg/day).
The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

Any other information on results incl. tables

Trimellitic anhydride chloride (TMAC): Summary of Micronucleus Data

Group	% PCE	% MN PCE	Statistic analysis WRS
	Mean  (SD)	Mean (SD)	P-value Significance
1 - Vehicle 0 mg/kg bw/day	49.00  (1.88)	0.10 (0.06)	N/A N/A
2- TMAC 266 mg/kg bw/day	49.70 (4.14)	0.13 (0.06)	0.1147 NS
3- TMAC 700 mg/kg bw/day	50.13 (6.21)	0.16 (0.08)	0.0996 NS
4- TMAC 1 400 mg/kg bw/day	42.70 (3.17)	0.12 (0.19)	0.8647 NS
5- Positive (EMS) 150 mg/kg bw/day	47.00 (2.82)	1.53 (0.35)	0.0119 *

EMS: Ethyl methanesulfonate / SD: Standard Deviation / WRS: Wilcoxon Rank Sum

PCE: Polychromatic erythrocytes / MN PCE: Micronucleated polychromatic erythrocytes

Applicant's summary and conclusion

Conclusions:

When tested in the micronucleus test conducted according to the OECD 474 guideline in rats exposed up to 1400 mg/kg bw/day (maximum tolerated dose), Trimellitic anhydride chloride (TMAC), did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow.

Executive summary:

Trimellitic anhydride chloride (TMAC) was tested for its potential to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of rats. The study was conducted according to the guideline OECD 474 and combined with the comet assay (according to the guideline OECD 489).

Male Sprague Dawley rats were treated by gavage once daily, for 3 days, at the actual dose levels of 266, 700 and 1400 mg/kg bw/day. Based on a range-finder experiment, the dose level of 1400 mg/kg bw/day was considered as being the MTD (maximum tolerated dose). The vehicle control group received the vehicle (corn oil) and the positive control group received Ethyl methanesulfonate (EMS) at 150 mg/kg bw/day.

Bone marrows from one femur were sampled at necropsy (Day 3, 48 hours, i.e. 3 hours after the last dosing).

The group mean vehicle control data (%MN PCE) was within the 95% reference ranges of the laboratory’s historical vehicle control data. The EMS positive control induced statistically significant increases in %MN PCE (over the current vehicle control group) that was comparable with the laboratory’s historical positive control data ranges. The assay was therefore accepted as valid.

There was a small reduction in group mean %PCE at 1400 mg/kg/day (-13%) that was observed in five animals. While the reduction was within the laboratory's historical control data, as it was observed in 5 out of 6 animals, it was considered potential evidence of bone marrow toxicity.

Animals treated with Trimellitic anhydride chloride (TMAC) at 266, 700 or 1400 mg/kg/day exhibited group mean %MN PCE frequencies that were similar to the vehicle control group mean and which fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control with no evidence of a dose response. Individual animal MN PCE frequencies were considered consistent with the laboratory's historical vehicle control animal distribution data. An isolated animal showed a %MN PCE value (0.50%) that exceeded the 95% reference range (0-0.33%); however, as this elevated value was not reproduced in any other animal in the high dose group and the group data was not statistically increased compared to the concurrent vehicle control group, this value was considered of no biological relevance.

It is concluded that under the conditions of this study, Trimellitic anhydride chloride (TMAC), did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow when tested up to 1400 mg/kg/day (an estimate of the maximum tolerated dose for this study).