4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Remarks:

In vivo Mammalian Alkaline Comet Assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

EC Number:

276-309-1

EC Name:

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

Cas Number:

72058-41-4

Molecular formula:

C11H18Cl2N4

IUPAC Name:

4,6-dichloro-N-(2,4,4-trimethylpentan-2-yl)-1,3,5-triazin-2-amine

Test material form:

solid

Details on test material:

Colour: light yellow

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SUQIAN UNITECH CO., LTD; Unitechem-20211231
- Purity: 99.29%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Samples were prepared according to a previous formulation study (Eurofins Munich Study No. 183815)

Test animals

Species:

rat

Strain:

other: Wistar rats, Crl: WI(Han) (Full Barrier)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 7-9 weeks old
- Weight at study initiation: 222-258g (first administration; males)
- Housing: The animals will be kept in groups of 2-3 animals / sex / group / cage in IVCs (type III H, polysulphone cages) on Altromin saw fibre bedding
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
According to a previous studies including a formulation study (Eurofins Munich Study No. 183815), the test item was prepared in Corn Oil and diluted prior to treatment. All animals received a single volume orally of 10 mL/kg bw. The solvent was chosen according to its relative non-toxicity for the animals. The prepared formulations were transferred to BSL Munich together with the control formulations under consideration of stability.

- Purity:

Details on exposure:

Test item and control formulations were prepared at Eurofins Munich (Eurofins Munich Study No.:STUGC22AA0720-2) and transferred to BSL Munich under consideration of the respective stability (Eurofins Munich Study No. 183815) to enable administration of all designated animals before expiration of the test item.

Duration of treatment / exposure:

2 consecutive days

Frequency of treatment:

0 and 24 hrs

Post exposure period:

Target organs are sampled at 4 hours after the last dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

LD

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

MD

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

HD

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control item ethylmethanesulfonate (EMS) was administered only once via gavage 4 hours before animal sacrifice at a dose of 250 mg/kg bw

Examinations

Tissues and cell types examined:

Isolation of primary hepatocytes:
A portion of the liver was minced with a pair of scissors to isolate the cells. The cell suspension was kept for not more than 15 seconds until bigger fragments of the liver settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

Isolation of small intestine, upper and lower part of colon cells:
The duodenum was flushed with a syringe filled with cold mincing buffer to wash out the food. The duodenum was cut open into two halves. One half of the duodenum was minced with a pair of scissors, the other one was kept for histopathology. The cell suspension was kept for not more than 15 seconds until bigger fragments settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

Isolation of glandular stomach cells:
The stomach was cut open and washed free of food using cold water. A portion of the glandular stomach was minced with a pair of scissors. The pieces were further crushed with a pestle to release single cells. The suspension was kept for less than 15 seconds to allow large clumps to settle. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

Isolation of germ cells from seminiferous tubules
The end of the epithelial capsule of the gonad was punctured to squeeze out the seminiferous tubules. Prepared cell pellets were used for preparing comet slides. Comet slides of the male gonads were stored at room temperature, under dry conditions and protected from light.

One part of the liver, glandular stomach, duodenum and one male gonad was preserved in 10% neutral-buffered formalin for histopathological evaluation

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A dose range-finding study on acute toxicity was performed with the same strain in both sexes to identify the maximum tolerated dose, defined as the dose inducing slight toxic effects at the end of the of the study period.

Under consideration of available data, a starting dose was chosen with the aim to elicit slight toxic symptoms. Based on acute oral toxicity data for female Wistar rats (Eurofins Munich / BSL Munich Study No. 183811), which showed moderate to strong signs of toxicity at a dose level of 2000 mg/kg bw, a limit dose of 1000 mg/kg bw was used in the dose range-finding experiment for female rats. As there is no information about the acute toxicity in male rats, a starting dose of 2000 mg/kg bw was used for male rats in the pre-experiment.

Due to the results obtained in the pre-experiment the highest dose for male rats was 1000 mg/kg bw and 500 mg/kg bw for female rats. After consultation of the sponsor, male rats were chosen for the main experiment as they showed the higher maximum tolerated dose.


DETAILS OF SLIDE PREPARATION:
Once single cells were obtained, the basic steps of the assay included:

Slide preparation:
The slides used were pre-coated with normal-melting agarose (NMA) and coded with a random number. A volume of 75 µL of cell suspension embedded in low-melting temperature agarose was placed on slides, which were covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue).

Lysis:
Cover slips were carefully removed and the slides incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow for the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed in purified water to remove residual detergent and salts.

Unwinding of DNA and electrophoresis:
Prior to electrophoresis, the slides were incubated in alkaline (pH > 13) electrophoresis solution for 20 min. An incubation period of 20 min was generally considered appropriate for alkali unwinding. After alkali unwinding, the single-stranded DNA was electrophoresed under alkaline conditions to enable the formation of DNA tails. The electrophoretic conditions were 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber in a random order.

Neutralisation and dehydration of slides:
After electrophoresis, the slides were neutralised by rinsing with neutralisation buffer three times for 5 min each. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards.

DNA staining:
Following dehydration, the cells were stained by applying 75 µL gel red staining solution on top of the slides and covering with a cover slip.

METHOD OF ANALYSIS:
Comet slides were analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software ‘Comet Assay IV’ (Perceptive Instrument, software version 2.1.2). The slides were coded so that the evaluator was not aware of which dose group was evaluated. Each slide was screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. The calculation of the different parameters was done automatically by the Comet Software, but the set front, middle and back lines of the comet might be adjusted manually if they were not set correctly automatically. All cells of the visual field were scored, except of e.g. overlapping cells, cells with an atypical nucleus, cells with a strong background or “hedgehogs” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail, were considered to be heavily damaged cells). Therefore, cells were classified into three categories scorable, non-scorable and “hedgehog”. To avoid artefacts only scorable cells and at least 150 cells per sample on two slides (75 cells per slide) were scored. The third back-up slide was scored in case of discordant results. The %-tail intensity is the parameter for evaluation and interpretation of DNA damage, and was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus.

Evaluation criteria:

Increases in DNA damage in the presence of a clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the % tail intensity in at least one dose group at a single sampling time in comparison with the negative control value.

Providing all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test,
- any of these results are outside the distribution of the historical negative control data

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).

To assess the biological relevance of a positive or equivocal result, information on cytotoxicity of the target tissue can be required. Where positive or equivocal findings are observed solely in the presence of a clear evidence for cytotoxicity, the study should be concluded as equivocal for genotoxicity unless there is enough information supporting a more definitive conclusion.

Statistics:

All slides, including those of positive and vehicle controls were independently coded and blinded before microscopic analysis. The median % tail intensity for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.

For each tissue type, the mean of the individual animal means was then determined to give a group mean % of tail intensity. Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p value was used as a limit in judging for significance levels in comparison with the corresponding vehicle control .

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Duodenum; at all doses

Toxicity:

yes

Remarks:

LD - piloerection MD - spontaneous activity, piloerection, prone position and hunched posture; four out of five male rats lost weight HD - spontaneous activity, piloerection, diarrhea and hunched posture; a reduction of body weight was noted in each male.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Glandular stomach; at all doses

Toxicity:

yes

Remarks:

LD - piloerection MD - spontaneous activity, piloerection, prone position and hunched posture; four out of five male rats lost weight HD - spontaneous activity, piloerection, diarrhea and hunched posture; a reduction of body weight was noted in each male.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Liver; at all doses

Toxicity:

yes

Remarks:

LD - piloerection MD - spontaneous activity, piloerection, prone position and hunched posture; four out of five male rats lost weight HD - spontaneous activity, piloerection, diarrhea and hunched posture; a reduction of body weight was noted in each male.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY

In the pre-experiment, one male rat received a single limit dose of 2000 mg/kg bw orally and showed severe signs of toxicity such as reduced spontaneous activity, prone position, piloerection, hunched posture and half eyes closed after the first administration of the test item. After the second administration of the test item, the same clinical signs were present but more severe in addition to moving the bedding.

Therefore, the dose was reduced to 1500 mg/kg bw and administered orally to one male rat. Most of the former effects were observed including reduced spontaneous activity, piloerection, hunched posture and half eyes closed after the first administration of the test item. After the second administration of the test item, the same clinical signs were present in addition to diarrhea.

Due to this observation, the dose was again reduced to 1000 mg/kg bw and administered orally to one male rat. After the first administration of the test item piloerection, hunched posture and reduced spontaneous activity were observed. Piloerection and diarrhea were still present before the second administration of the test item. During the observation period of 4h, reduced spontaneous activity, diarrhea and hunched posture were additionally observed. Due to the results obtained, two additional male rats were treated with a dose of 1000 mg/kg bw. Reduced spontaneous activity, piloerection, prone position and diarrhea were observed after the first administration of the test item. During the observation period of 4h, reduced spontaneous activity, diarrhea and piloerection were observed (see Appendix 2: Study-related Toxicity Data; Table 10).
In the pre-experiment, one female rat received a single dose of 1000 mg/kg bw orally and showed clinical signs such as reduced spontaneous activity, hunched posture and piloerection after the first administration of the test item. After the second administration of the test item, piloerection was observed. Therefore, two additional female rats were dosed with 1000 mg/kg bw. Both female rats showed reduced spontaneous activity and piloerection after the first administration of the test item. After the second administration of the test item, the females showed more severe clinical signs, such as reduced spontaneous activity, piloerection, hunched posture, diarrhea and half eyes closed (only one female; (see Appendix 2: Study-related Toxicity Data; Table 10).

Therefore, the amount of the test item was reduced to 500 mg/kg bw/d and administered orally to one female rat. After the first administration of the test item piloerection and reduced spontaneous activity were observed. Piloerection and diarrhea were present before the second administration of the test item. During the observation period of 4h, reduced spontaneous activity, hunched posture and piloerection were observed. The toxicity signs noted in female rats after the application of 500 mg/kg bw/d were classified as slight to moderate toxic effects, therefore two additional female rats received the same dosing to confirm the concentration of 500 mg/kg bw/d as HD for female rats. Piloerection and reduced spontaneous activity were observed after the first administration of the test item and were also present after the second administration of the test item.

Due to the results obtained in the pre-experiment the highest dose for male rats was 1000 mg/kg bw and 500 mg/kg bw for female rats. After consultation of the sponsor, male rats were chosen for the main experiment as they showed the higher maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY

Clinical signs and body weight
All animals treated with the highest dose (HD) showed slight to moderate toxic effects such as reduction of spontaneous activity, piloerection, diarrhea and hunched posture after the first and second application (Appendix 2: Study-related Toxicity Data; Table 12). Animals treated with a dose of 500 mg/kg bw showed spontaneous activity, piloerection, prone position and hunched posture after the first and second application (see Appendix 2: Study-related Toxicity Data; Table 11). Rats treated with 200 mg/kg bw (LD) showed piloerection after the second administration of the test material.

Three out of five male rats in the vehicle control group (corn oil) lost weight at the end of the experiment. A reduction of body weight was noted in each male animal of the highest dose group (1000 mg/kg bw) for male rats at the end of the experiment. At the lower concentrations tested, four out of five male rats in the MD group (500 mg/kg bw) lost weight. All male rats in the lowest does group (200 mg/kg bw) gained weight by the end of the experiment.

GENOTOXICITY
Liver
The group mean value obtained in the main experiment for the vehicle control was 1.03 % in the case of male liver cells (Table 3). The tail intensity of the LD (200 mg/kg bw; 1.18 %), MD (500 mg/kg bw; 1.08 %) and HD (1000 mg/kg bw; 0.87 %) were within the historic control limits of the testing facility (0.07– 3.82%; 16.1 Appendix 1: Laboratory Historical Control Data). No significant increase compared to the vehicle control (corn oil) was noted for any dose group evaluated for male liver cells (Table 6). No concentration-dependency was noted for the tail intensities in male cells of the liver (Table 7). Ethyl methanesulfonate (EMS) as an appropriate DNA damaging agent (250 mg/kg bw) was used as positive control. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 11.88% derived from male liver cells. This demonstrates the validity of the assay.

Glandular stomach
In glandular stomach cells, the group mean value obtained for the vehicle control was 3.26% (Table 4). The tail intensity of the dose groups at 200 mg/kg bw (LD; 4.61%), 500 mg/kg bw (MD; 3.70%) and 1000 mg/kg bw (HD; 3.07%) were within the historic vehicle control range of the testing facility (1.30- 5.46%, 16.1 Appendix 1: Laboratory Historical Control Data). No significant increases compared to the vehicle control (corn oil) was noted for any dose group evaluated in male glandular stomach cells (Table 6). No concentration-dependency was noted for the tail intensities in male cells of the glandular stomach (Table 7). Ethyl methanesulfonate (EMS) as an appropriate DNA damaging agent (250 mg/kg bw) was used as positive control. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 12.19% derived from male glandular stomach cells

Duodenum
In the duodenum, the group mean value obtained for the negative control was 2.39% (Table 5). The group mean tail intensities of the test item concentrations 200 mg/kg bw (LD; 2.15%), 500 mg/kg bw (MD; 3.95%) and 1000 mg/kg bw (HD; 2.39%) were within the range of the historic vehicle control with tail intensities 0.89- 4.06% (16.1 Appendix 1: Laboratory Historical Control Data). No significant increases compared to the vehicle control (corn oil) was noted for any dose group evaluated in male duodenum cells (Table 6). No concentration-dependency was noted for the tail intensities in male cells of the duodenum (Table 7). Ethyl methanesulfonate (EMS) as an appropriate DNA damaging agent (250 mg/kg bw) was used as positive control. In vivo treatment with EMS revealed a significant increase in DNA damage with tail intensity values of 11.08% derived from male duodenum cells. This demonstrates the validity of the assay.

No increase of hedgehogs was detected in all organs evaluated (see Table 13, Table 14 and Table 15).

STATISTICS
Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p value was used as a limit in judging for significance levels in comparison with the corresponding vehicle control.
A statistically significant increase was determined for the positive control group of each organ. No significances compared to the vehicle control were determined in any of the dose groups of each organ (Table 6).

For the determination of a statistically significant concentration-related increase, the mean values of each animal per dose group were evaluated with a linear trend test at the 5 % level (p<0.05). The p-value was used as a limit in judging for significance levels.
No concentration-related increase was noted in any of the analysed organs in male rats (Table 7).

As the results in all 3 tissues were clearly negative and target tissue exposure was confirmed, the male gonad slides were not evaluated.

Any other information on results incl. tables

Less than 150 cells were scored for one male in the HD group (animal 37 (149 cells) in glandular stomach cells as not enough cells were present for evaluation (see Table 26). Nevertheless, this did not affect the validity of the assay, as still 149 cells (animal 2) were scored and these values were in the same range as the tail intensities of the animals 1, 3,4 and 5 of the same dose group.

 

Applicant's summary and conclusion

Conclusions:

In an in vivo Mammalian Alkaline Comet Assay of liver, glandular stomach and duodenum cells in male Wistar rats, the test item did not induce biologically relevant DNA-strand breaks in any of the tissues evaluated. The result indicates no adverse effect of the test item on the DNA of liver, glandular stomach and duodenum cells after oral administration to rats.

Executive summary:

In a Wistar rat comet assay, groups of 5 male rats were treated by oral gavage with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) in corn oil at doses of 200, 500 and 1000 mg/kg bw. The animals were dosed on 2 consecutive days (0, 24 ± 1 hrs) and samples of the liver, glandular stomach, duodenum and male gonads were removed four hours after the last administration of the test item. The positive control was ethyl methanesulfonate (250 mg/kg bw). A total of 150 cells/animal/tissue (75 cells per slide) were evaluated and DNA migration during electrophoresis was determined and expressed as % tail intensity. Systemic toxicity was evaluated via clinical signs and body weight changes.

 

All animals treated with the highest dose (HD) showed slight to moderate toxic effects such as reduction of spontaneous activity, piloerection, diarrhea and hunched posture after the first and second application. Animals treated with a dose of 500 mg/kg bw showed spontaneous activity, piloerection, prone position and hunched posture after the first and second application. Rats treated with 200 mg/kg bw (LD) showed piloerection after the second administration of the test material. A reduction of body weight was noted in each male animal of the highest dose group (1000 mg/kg bw) for male rats at the end of the experiment. At the lower concentrations tested, four out of five male rats in the MD group (500 mg/kg bw) lost weight. All male rats in the lowest does group (200 mg/kg bw) gained weight by the end of the experiment. Therefore, the highest dose (1000 mg/kg bw) showed clinical signs that were strong enough to demonstrate systemic exposure without evidence of study-limiting toxicity.

 

The group mean value obtained in the main experiment for the vehicle control was 1.03 % in the case of male liver cells. The tail intensity of the LD (200 mg/kg bw; 1.18 %), MD (500 mg/kg bw; 1.08 %) and HD (1000 mg/kg bw; 0.87 %) were within the historic control limits of the testing facility (0.07– 3.82%). No significant increase compared to the vehicle control (corn oil) was noted for any dose group evaluated for male liver cells. No concentration-dependency was noted for the tail intensities in male cells of the liver. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 11.88% derived from male liver cells.

 

In glandular stomach cells, the group mean value obtained for the vehicle control was 3.26%. The tail intensity of the dose groups at 200 mg/kg bw (LD; 4.61%), 500 mg/kg bw (MD; 3.70%) and 1000 mg/kg bw (HD; 3.07%) were within the historic vehicle control range of the testing facility (1.30- 5.46%). No significant increases compared to the vehicle control (corn oil) was noted for any dose group evaluated in male glandular stomach cells. No concentration-dependency was noted for the tail intensities in male cells of the glandular stomach. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 12.19% derived from male glandular stomach cells.

 

In the duodenum, the group mean value obtained for the negative control was 2.39%. The group mean tail intensities of the test item concentrations 200 mg/kg bw (LD; 2.15%), 500 mg/kg bw (MD; 3.95%) and 1000 mg/kg bw (HD; 2.39%) were within the range of the historic vehicle control with tail intensities 0.89- 4.06%. No significant increases compared to the vehicle control (corn oil) was noted for any dose group evaluated in male duodenum cells. No concentration-dependency was noted for the tail intensities in male cells of the duodenum. In vivo treatment with EMS revealed a significant increase in DNA damage with tail intensity values of 11.08% derived from male duodenum cells.

 

No increase of hedgehogs was detected in all organs evaluated. No concentration-related increase was noted in any of the analysed organs in male rats. As the results in all 3 tissues were clearly negative and target tissue exposure was confirmed, the male gonad slides were not evaluated.

 

Therefore, the test item did not induce biologically relevant DNA-strand breaks in any of the tissues evaluated. The result indicates no adverse effect of the test item on the DNA of liver, glandular stomach and duodenum cells after oral administration to rats.