Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and Ethanol, 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-

Genetic toxicity: in vivo

Administrative data

Endpoint:

genetic toxicity in vivo, other

Remarks:

DNA strand breaks in liver, stomach and duodenum

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2016 - February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted September 26, 2014 (A new version of the guideline has been adopted by OECD. The study procedures described in this report are also in compliance with this new guideline:
Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing of Chemicals, Guideline No. 489: In Vivo Mammalian Alkaline Comet Assay (adopted July 29, 2016).

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(4-methylphenyl)imino]bisethanol

EC Number:

221-359-1

EC Name:

2,2'-[(4-methylphenyl)imino]bisethanol

Cas Number:

3077-12-1

Molecular formula:

C11H17NO2

IUPAC Name:

2-[(2-hydroxyethyl)(4-methylphenyl)amino]ethan-1-ol

Constituent 2

Reference substance name:

2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethan-1-ol

Cas Number:

878391-30-1

Molecular formula:

C13H21NO3

IUPAC Name:

2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethan-1-ol

Constituent 3

Reference substance name:

Higher ethoxylated p-toluidine derivative-1

IUPAC Name:

Higher ethoxylated p-toluidine derivative-1

Constituent 4

Reference substance name:

Higher ethoxylated p-toluidine derivative-2

IUPAC Name:

Higher ethoxylated p-toluidine derivative-2

impurity 1

Test material form:

liquid: viscous

Details on test material:

- Physical appearance: Clear slightly yellowish to brown viscous liquid
- Storage conditions: At room temperature

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: 149 ± 7.9 g (range 131 – 162 g) in experiment 1 and 149 ± 7.8 g (range 137 – 168 g) in the repeat experiment.
- Assigned to test groups randomly: yes
- Fasting period before study: no (A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat)

- Housing: Group housing of maximum 5 animals per sex in labeled Macrolon cages (type MIV) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): free access to tap-water
- Acclimation period: at least 6 days before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2– 21.4
- Humidity (%): 30 - 73
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 May 2016 To: 16 February 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Stable for 6 hours in the range of 20-200 mg/mL in propylene glycol.
No correction was made for the purity/composition of the test compound.
Accelerator (PT 25E or PT 25E/2) was dissolved in propylene glycol. The specific gravity of propylene glycol is 1.036 g/ml. Accelerator (PT 25E or PT 25E/2) concentrations were treated with ultra-sonic waves to obtain a homogeneous solution (sonication: max. time
12 min, max. temp 28°C in experiment 1 and max. time 14 min, max. temp 32°C in the repeat experiment). Accelerator (PT 25E or PT 25E/2) concentrations were dosed within 3 hours after preparation in experiment 1 and within 2 hours in the repeat experiment.

Duration of treatment / exposure:

three consecutive days

Post exposure period:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

187.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

375 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

at least 5 males per treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate
- Route of administration: oral
- Doses / concentrations: 10 mL/kg bw; 200 mg/kg bw

Examinations

Tissues and cell types examined:

liver, stomach and duodenum

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Selection of an adequate dose range for the in vivo Comet main test was based on a dose range finding study. The test procedure and conditions were similar to those applied in the main test. In a sub-acute toxicity study with the test substance no gender difference was observed. Based on these data the dose range finding study and the main studies were conducted in males only.
Two dose groups, one comprising of 1 male (600 mg/kg bw) and the other comprising of 3 males (750 mg/kg bw) were dosed for three consecutive days (once daily) with Accelerator (PT 25E or PT 25E/2). The group comprised of 3 males were dosed with the highest concentration that was used for the main study. The observation period after dosing was one to 3 days. During this period mortality and physical condition were recorded at least once a day.
The dose range study was started with a dose of 600 mg/kg bw. This dose was selected for ethical reasons and based on the results of the 28-day repeated dose toxicity study in rat.



TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Approximately 3-4 hours after the third treatment with the test compound or vehicle and second treatment with EMS liver (first experiment only), stomach and duodenum tissue was collected/isolated and examined for DNA damage with the alkaline Comet assay.

DETAILS OF SLIDE PREPARATION:To 20 µL of the cell suspension, 280 µL melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added. The cells were mixed with the LMAgarose and 50 µL was layered on a precoated Comet slide (Trevigen) in duplicate. Three slides per tissue were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for at least 10 minutes (actual time 15-24 minutes in experiment 1 and 16-23 minutes in the repeat experiment) in the refrigerator in the dark until a clear ring appeared at the edge of the Comet slide area.
The cells on the slides were overnight (approximately 16-18 h in both experiments) immersed in prechilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4) for approximately 5 minutes. The slides were then placed in freshly prepared alkaline solution for 25-56 minutes in experiment 1 and for 20-32 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1 Volt/cm. The electrophoresis was performed for 30 minutes under constant cooling (actual temperature 5.0 – 7.5°C in experiment 1 and 4.5 – 6.0 °C). After completion of electrophoresis, the slides were immersed/rinsed in the neutralization buffer. The slides were subsequently immersed for approximately 5 minutes in absolut ethanol (≥99.6%) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark.

METHOD OF ANALYSIS:
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample. On a few slides, one of the agorose circles was damaged, therefore an agarose circle from the second backup slide was used for scoring.
The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.

OTHER:One experiment was perfomed for liver and two experiments were performed for glandular stomach and duodenum since the first experiment did not pass the acceptance criteria for the control group.

Evaluation criteria:

A test compound is considered positive in the Comet assay (in a tissue) if the following criteria are met:
It induces a biologically as well as a statistically significant (Dunnett’s test, one-sided,
p < 0.05) dose-dependent increase in percentage Tail Intensity. In case of other non-dose-dependent significant increases the data interpretation will be on a case by case base.

A test compound is considered as negative in the Comet assay (in a tissue) if the following criteria are met:
None of the tested concentrations show a statistically significant (Dunnett’s test, one-
sided, p < 0.05) dose-dependent increase in percentage Tail Intensity.

Data was normally distributed thus no transformation (y = 1/y) of the data was necessary. In addition no Cochran Armitage trend test (p < 0.05) was performed.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis.

Statistics:

Dunnett’s test, one-sided; ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

high dose animals: lethargic and tremors first hour after dosing; one animal died after the third dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 600 (1 male) and 750 (3 males) mg/kg bw/day
- Clinical signs of toxicity in test animals: The dose range finding study was started with a dose of 600 mg/kg. At 600 mg/kg bw/day the effects were minor: the male animal was only lethargic after the first dose and recovered thereafter and no treatment related clinical signs were observed after the second and third dose. It was therefore concluded that a dose of 600 mg/kg bw/day was too low as maximum concentration in the main assay.
Subsequently three male animals were dosed with 750 mg/kg bw/day. The following clinical signs were observed during the duration of the study (days 1-3): lethargy, hunched posture, rough coat and tremors. One animal died after the first dosing with 750 mg/kg bw/day.


RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: Based on the results of the dose range finding study dose levels of 187.5, 375 and 750 mg/kg body weight were selected as appropriate doses for the Comet main test.
Five male animals were used in each treatment group. Three additional male animals, treated with 750 mg Accelerator (PT 25E or PT 25E/2)/kg body weight, were used to correct for possible deaths.
After treatment, single cell suspensions were prepared from the liver, stomach and duodenum tissue. The viability of one single cell suspension per tissue per group was assessed by using trypan blue. The viability of the single suspension was 90-100% in experiment 1 and 97 – 100% in the repeat experiment. The viability was assessed for one animal per group.
- Statistical evaluation:
No statistically significant increase in the mean Tail Intensity (%) was observed in liver cells, stomach cells and duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals.

Any other information on results incl. tables

The concentrations analysed in the formulations were considered in agreement with target concentrations (i.e. mean accuracies between 102% and 106%). No test item was detected in the vehicle control samples.

Liver

The mean Tail Intensity (%) in liver cells of vehicle treated male rats was 4.66 ± 1.60% (Mean± SD). The mean vehicle control Tail Intensity was within the historical control data range. The positive control EMS showed a mean Tail Intensity of 98.05 ± 0.48% (Mean± SD,21-fold induction; p<0.001). Overall it was concluded that the comet assay in liver was valid.

Stomach (Experiment 1)

No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals.

The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 78.50 ± 7.92% (Mean± SD). The mean vehicle control Tail intensity was above the historical control data range. The positive control EMS showed a mean Tail Intensity of 99.01 ± 0.83% (Mean± SD,1.3-fold induction; p<0.001).

Overall the acceptability criteria of the test were not met; the percentage tail intensity of the solvent control outside the laboratory historical control data range. Therefore although no increase in Tail intensity (%) is observed no conclusion can be drawn about the potential DNA damaging properties of Accelerator (PT 25E or PT 25E/2) in glandular stomach. A repeat experiment was performed.

Stomach (Repeat)

No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 70.33 ± 3.42% (Mean± SD). The mean vehicle control Tail intensity was within the historical control data range. The positive control EMS showed a mean Tail Intensity of 95.47 ± 0.62% (Mean± SD,1.4-fold statistically significant induction; Studentsttest p<0.001). Overall it was concluded that the comet assay (repeat) in stomach was valid.

Duodenum (Experiment 1)

No statistically significant increase in the mean Tail Intensity (%) was observed in duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals.

The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 74.13 ± 11.25% (Mean± SD). The mean vehicle control Tail intensity was above the historical control data range.The positive control EMS showed a mean Tail Intensity of 99.58 ± 0.25% (Mean± SD,1.3-fold induction; p<0.001).

Overall the acceptability criteria of the test were not met; the percentage tail intensity of the solvent control is outside the laboratory historical control data range. Therefore although no increase in Tail intensity (%) is observed no conclusion can be drawn about the potential DNA damaging properties of Accelerator (PT 25E or PT 25E/2) in duodenum. A repeat experiment was performed.

Duodenum (Repeat)

No statistically significant increase in the mean Tail Intensity (%) was observed in duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals.

The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 55.34 ± 15.40% (Mean± SD). The mean vehicle control Tail intensity was within the historical control data range.The positive control EMS showed a mean Tail Intensity of 97.03 ± 0.51% (Mean± SD,1.8-fold induction; p<0.001). Overall it was concluded that the comet assay (repeat) in duodenum was valid.

Overall the results in liver, stomach and duodenum show that there is no statistically significant induction and thus also no trend of induction in the Tail Intensity (%) in Accelerator (PT 25E or PT 25E/2)-treated male animals.

Applicant's summary and conclusion

Conclusions:

It is concluded that the comet assay in liver, duodenum and glandular stomach were valid and Accelerator (PT 25E or PT 25E/2) does not provoke DNA damage in the Comet assay up to and including a dose of 750 mg/kg bw/day (the maximum tolerated dose in accordance with current regulatory guidelines).

Executive summary:

Accelerator (PT 25E or PT 25E/2) was tested in the alkaline in vivo Comet assay in male rats, to evaluate its potential genotoxic effect in liver, glandular stomach and duodenum cells. The study procedures described in this report were based on the most recent OECD guidelines. The test item was dissolved in propylene glycol. Formulation analysis was performed to determine the accuracy of preparation of the test substance in formulations. The concentrations analysed in the formulations were in agreement with target concentrations (i.e. mean accuracies between 102% and 106%) . No test item was detected in the vehicle control samples. Based on the dose range finding study a dose of 750 mg/kg bw/day was selected as highest dose for the main study (maximum tolerated dose).

One experiment was perfomed for liver and two experiments were performed for glandular stomach and duodenum since the first experiment did not pass the acceptance criteria for the control group.

In the main studies, groups of 5 male animals were dosed once daily via oral gavage with vehicle or with 187.5, 375 and 750 mg Accelerator (PT 25E or PT 25E/2) per kg body weight for three consecutive days. Three additional satellite animals were treated with the highest test concentration to replace possible deads. A positive control group (5 male rats) for the comet assays was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight. The following treatment related clinical signs were observed the groups treated with 750 mg Accelerator (PT 25E or PT 25E/2)/kg body weight in experiment 1: lethargy, tremors, hunched posture, quick breathing, convulsions and ventral recumbency. One animal died within 15 minutes after the third dose. This animal was replaced by a satellite animal. The treatment related clinical signs in the repeat assay were similar. The following treatment related clinical signs were observed in the groups treated with 750 mg Accelerator (PT 25E or PT 25E/2)/kg body weight in the repat assay: lethargy, tremors, ataxia and ventral recumbency. Approximately 3-4 hours after the second dose of EMS and third dose of the vehicle or Accelerator (PT 25E or PT 25E/2), liver, glandular stomach, and duodenum tissue were collected. Single cell suspensions were made followed by Comet slide preparation and assessment of Tail Intensity (%). No biologically relevant increase in the mean Tail Intensity (%) was observed in liver cells of Accelerator (PT 25E or PT 25E/2) treated male animals compared to the vehicle treated animals (3.23%, 3.55% and 2.62% at 187.5 mg/kg bw, 375 mg/kg bw and 750 mg/kg bw, respectively) . The mean Tail Intensity (%) in liver cells of vehicle treated male rats was 4.66 ± 1.60% (Mean± SD). The mean Tail Intensity was within the historical control data range for the vehicle control group. The positive control EMS showed a mean Tail Intensity of 98.05 ± 0.48% (Mean± SD,21-fold induction; statistically significant according to Studentsttest p<0.001). Overall it was concluded that the comet assay in liver was valid.

No statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach and duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals in experiment 1.

The vehicle control Tail intensities in glandular stomach and duodenum, were above the historical control data range in experiment 1. Therefore the acceptability criteria of these assays were not met; the percentage tail intensity of the solvent control is outside the laboratory historical control data range.

The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 78.50 ± 7.92% (Mean± SD) in experiment 1. The positive control EMS showed a mean Tail Intensity of 99.01 ± 0.83% (Mean± SD,1.3-fold induction; statistically significant according to Studentsttest p<0.001).

The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 74.13 ± 11.25% (Mean± SD). The positive control EMS showed a mean Tail Intensity of 99.58 ± 0.25% (Mean± SD,1.3-fold induction; statistically significant according to Studentsttest p<0.001). Although no increase in Tail intensity (%) in glandular stomach and duodenum was observed for any treatment group in experiment 1 (82.53%, 73.25% and 65.93% for stomach and 80.78%, 55.08% and 41.77% for duodenum at 187.5 mg/kg, 375 mg/kg and 750 mg/kg, respectively), no conclusion could be drawn about the potential DNA damaging properties of Accelerator (PT 25E or PT 25E/2) in duodenum and glandular stomach since the acceptance criteria for the control group were not passed. The mean vehicle control Tail intensity was above the historical control data range in stomach and duodenum.

Therefore a repeat experiment was performed for glandular stomach and duodenum. No statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach (70.33%) and duodenum cells (55.34%) of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals in the repeat experiment.

The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 70.33 ± 3.42% (Mean± SD). The mean vehicle control Tail intensity was within the historical control data range. The positive control EMS showed a mean Tail Intensity of 95.47 ± 0.62% (Mean± SD,1.4-fold induction; p<0.001). Overall it was concluded that the comet assay (repeat) in glandular stomach was valid.

The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 55.34 ± 15.40% (Mean± SD). The mean vehicle control Tail intensity was within the historical control data range. The positive control EMS showed a mean Tail Intensity of 97.03 ± 0.51% (Mean± SD,1.8-fold induction). Overall it was concluded that the comet assay (repeat) in duodenum was valid.

Overall the results in liver, stomach and duodenum show that there is no statistically significant induction and thus also no trend of induction in the Tail Intensity (%) in Accelerator (PT 25E or PT 25E/2)-treated male animals.

It is concluded that the comet assay in liver, duodenum and glandular stomach is valid and Accelerator (PT 25E or PT 25E/2) does not provoke DNA damage in the Comet assay up to and including a dose of 750 mg/kg bw (the maximum tolerated dose in accordance with current regulatory guidelines).