Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols

Endpoint summary

Description of key information

Subacute (28-day) repeated dose toxicity (OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, rat): NOAEL ≥ 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 2018 - 30 Sept 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Rathausgasse 4, 91126 Schwabach, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

recommended by guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12-13 weeks
- Weight at study initiation: males: 345 - 420 g; females: 217 - 277 g
- Housing: in groups of 5 animals/sex/cage, type IV polysulphone cages or double decker IVC cages during premating period for males and females and during post-mating period for males; males and females co-housed during mating period in ratio 1:1; females housed in type III H polysulphone cages after confirmation of mating; Altromin saw fibre used as bedding in each cage
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: 18 - 23 July 2018

DETAILS OF FOOD AND WATER QUALITY: Certificates of food, water and bedding are filed for two years at the testing facility

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 7 Aug 2018 To: 29 Sept 2018

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in corn oil. The test item was weighed into a tared plastic vial on a suitable precision balance. The appropriate amount of vehicle was added to obtain the final concentration of the test item. A solution with the vehicle was prepared by subjecting the test item-vehicle suspension for approximately 60 min at 40 °C to an ultrasonic bath and 45 min stirring using a heating plate at 40 °C. To keep formulations fluid during the daily administration, they were kept under magnetic stirring and warm (≤ 40 °C) using a heating plate. Based on the results of stability testing, the test item formulations were prepared at least once every 7 days. The prepared formulation was stored protected from light and at room temperature.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Consultation with the sponsor based on the test item’s characteristics and testing guideline
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): MKCF8882, MKCD1021
- Expiry dates: 12 November 2018 (MKCF8882), 24 November 2018 (MKCD1021)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups. As the test item was not shown to be homogenous, samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) (40 samples). The samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 180922) and until then stored under appropriate conditions based on available stability data.

Duration of treatment / exposure:

Males: 28 days (14 days pre-mating period, 14 days mating period)
Females: 63 days (14 days premating period, 14 days mating period, approx. 22 days through gestation period until post-natal day 12)

Frequency of treatment:

7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a dose range finding study (BSL Munich Study No. 181975) and in consultation with the sponsor. The animals in the dose range finding study showed no mortality and only slight test item-related clinicial signs. Test item-treated animals showed no gross difference in body weight development and no difference in food consumption compared to the control animals. The test item-treatment was found to have no effects on reproductive indices as well as pup-related parameters. Thus, the dose levels proposed to be used in the higher tier study are 100, 300, and 1000 mg/kg/day.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before first exposure, at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 h of parturition (day 0 post-partum), on post-natal day (PND) 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination.

FOOD CONSUMPTION: YES
- Time schedule: on the same days as body weight measurements, not measured during mating period in males and females and post-mating period in males

FOOD EFFICIENCY: NO

OPHTHALMOSCOPIC EXAMINATION: Yes
No further details reported.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment prior to or as part of the sacrifice of the animals by using anesthesia (ketamine/xylazine).
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters summarised in table 1 were examined. Refer to 'Any other information on materials and methods incl. tables' below.
- Details on coagulation:
- Time schedule for examinations: at the end of the treatment prior to or as part of the sacrifice of the animals
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) of each group
- Parameters tested: prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment prior to or as part of the sacrifice of the animals
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) of each group
- Parameters summarised in table 2 were examined. Refer to 'Any other information on materials and methods incl. tables' below.

URINALYSIS: Yes
- Time schedule for collection of urine: 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: No
- Animals fasted: Not specified
- Parameters summarised in table 3 were examined. Refer to 'Any other information on materials and methods incl. tables' below.

NEUROBEHAVIOURAL EXAMINATION / / FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Time schedule for examinations: males: in the week before first treatment and during the last week of treatment in 5 randomly selected males; females: during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights: The wet weight of organs (table 4, refer to 'Any other information on materials and methods incl. tables' below) of 5 randomly selected male and female animals (only lactating females were evaluated) from each group were recorded. Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals.
- Further details: All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY: Yes
- Parameters summarised in table 5 were examined. Refer to 'Any other information on materials and methods incl. tables' below.
- Further details: Full histopathology was carried out on the preserved organs and tissues of the selected animals of the control and high dose groups. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test.
Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
Statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p < 0.05 was considered as statistically significant).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: Females during first premating week: absolute body weight increased to a level of approx. 2 % above controls; non adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

MORTALITY:
No mortality occurred in the during the treatment period of this study.

CLINICAL OBSERVATIONS:
Moving the bedding was seen in all female animals of the 300 mg/kg bw/day and 1000 mg/kg bw/day groups transiently during gestation and postnatal periods. A single 100 mg/kg bw/day female was observed moving the bedding on few gestation days. This clinical sign was observed immediately after the dose administration and therefore is considered to be a sign of a local reaction to the test item rather than a systemic adverse effect, and is not toxicologically relevant. Moving the bedding was not observed in male animals of the dose groups. Single or occasional findings that are assumed to be related to the gavage technique or that are considered incidental were: abnormal breathing, lacrymation, regurgitation of the test item, increased salivation, diarrhea, transient piloerection, scratches, crust or wounds. Hairless areas occurred in a few animals of all groups. This is usually observed in this strain and is not assumed to be toxicologically relevant.

FUNCTIONAL OBSERVATIONS:
In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.

OPHTHALMOLOGY:
No opthalmoscopic alterations were seen at the end of the treatment period in any of the animals.

BODY WEIGHT DEVELOPMENT:
A statistically significant higher body weight gain was observed in females in the 1000 mg/kg bw/day group during the first premating week. This is not considered to be toxicologically relevant as the absolute body weight of these animals only increased to a level of approx. 2% above controls. Body weights of male animals were in the normal range of variation throughout the treatment period of the study and there were no statistically significant differences between dose groups and controls.

FOOD CONSUMPTION:
The test item had no toxicologically relevant effect on food consumption in male and female animals of this study. Slightly and statistically significant higher food intake was seen in female animals of the 1000 mg/kg bw/day group during the first 2 weeks of gestation and the last week of the postnatal period (Day 4 to 13). This is not considered to be an adverse effect.

HAEMATOLOGY AND COAGULATION:
A statistically significant increased rate of large unstained cells (78% above controls) was found in the blood of females of the 1000 mg/kg bw/day group. Large unstained cells are assumed to be progenitor cells of the bone marrow released into circulation at a low rate. In absence of changes in white and red blood cells and histopathological findings in bone marrow, this is assumed not to be of biological relevance. Moreover, all values were in the range of historical control data. In addition, there were no significant differences in haematological parameters between dose groups and control group and all were within the normal range of variation. There was no statistically significant effect of the test item on coagulation parameters (PT, aPTT) determined at the end of the treatment period.

CLINICAL BIOCHEMISTRY:
There was no biologically relevant effect of the test item on clinical biochemistry parameters determined at the end of the treatment period of this study. A slight, but statistically significant higher serum urea level (26% above controls) found in female animals of the 300 mg/kg bw/day group was found. Due to the slightness and the lack of a dose-dependency this is not considered to be of toxicological relevance. Moreover, this did not coincide with any abnormalities observed in the renal system.

URINALYSIS:
The urinalysis performed in male and female animals sacrificed at the end of treatment period showed no test item treatment-related effect in all treatment groups. All urinary parameters were in the normal range of variation.

PATHOLOGY:
Only single or occasional macroscopic findings were noted in the groups during necropsy of the animals. These are assumed to be incidental.

ORGAN WEIGHTS:
There were no statistically significant differences in organ weights between dosed and control animals. A tendency towards slightly higher absolute and relative (to body weight) adrenal weight in male (15% above controls, respectively) and female animals (23 and 27% above controls, respectively) of the 1000 mg/kg bw/day group is possibly related to a secondary stress response caused by gavaging of the test item formulation. A high mean thymus weight found in females of the 100 mg/kg bw/day group is caused by a markedly higher thymus weight of animal no. 54. There were no relevant findings from the clinical pathology or histopathology. A tendency towards higher ovary weight in all dose groups (between 26 and 29% above controls) is not considered to be toxicologically relevant as there were no corresponding histopathological findings.

HISTOPATHOLOGY:
No test item related changes were noted. All recorded findings were deemed to be incidental or fell within the range of background alterations that may be recorded in Wistar rats. There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. No treatment-related effects on the testicular histomorphology were observed. No treatment-related effect on interstitial cell structure was noticed.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed up to and including the highest dose tested

Key result

Critical effects observed:

no

Tables summarising individual animal data are attached as pdf documents to the technical dossier.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6.1, of Regulation (EC) No. 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The repeated dose toxicity following oral exposure of reaction product of 1,3,5-triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols was investigated in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422 and under GLP conditions (Allingham, 2019). The test item was administered by oral gavage in doses of 100, 300 and 1000 mg/kg bw/day, 7 days/week, to male and female Wistar rats. Females were treated for a period of up to 63 days and males were dosed until the minimum total dosing period of 28 days was completed. Animals of the control group were handled identically to the dose groups but received the vehicle, corn oil. The four groups comprised 10 male and 10 female rats each. During the administration period, the animals were observed each day for signs of toxicity. At the end of the test period, all animals were sacrificed and a macroscopic evaluation was performed. Body weight and food consumption were recorded on gestation day 0, 7, 14 and 20, and within 24 hours of parturition (day 0 post-partum), and on post-natal day (PND) 4, PND 9 and PND 13. Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five randomly selected males and 5 females from each group. Urinalysis was performed on samples collected at terminal sacrifice from 5 randomly selected males and females (only lactating females were evaluated) from each group. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavioural observations were performed in the week before the treatment on all animals and in the last week of treatment on 5 randomly selected males and 5 females of each group. Ophthalmological investigations were performed at the of the observation period.

No test item-related mortality or clinical signs of systemic toxicity were observed during daily and detailed weekly observations. Immediately after the dose administration, female animals of the mid-dose and high-dose groups were observed moving the bedding. This is considered to be a sign of a transient local reaction to the test item. At the end of the treatment period there were no signs of a neuro-behavioural effect of the test item in the functional observation battery tests. There were no ophthalmological effects of the test item in this study. The test item had no toxicologically relevant effect on body weight or body weight gain, or on food consumption in both sexes. There was no effect of the test item on haematological, clinical biochemistry and coagulation parameters determined at the end of the treatment period. There was no treatment-related effect on urinary parameters in this study. No test item-related macroscopic findings were noted in the groups during necropsy of the animals. There were no toxicologically significant differences organ weights between dosed and control animals. No treatment-related changes were observed during the histopathological evaluation.

Based on these results, the No-Observed-Adverse-Effect-Level (NOAEL) for general toxicity is determined to be ≥ 1000 mg/kg bw/day in male and female rats.

Justification for classification or non-classification

The available data on repeated dose toxicity of the registered substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP), and are, therefore, conclusive but not sufficient for classification.