Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with formaldehyde, oligomeric reaction products with acetone and diphenylamine in rats by oral gavage.

Parental NOAEL: 50 mg/kg (based on adverse microscopic alterations in the liver and associated changes in clinical pathology parameters in both sexes, and reduced body weight gain and food consumption during lactation in females at the higher dose levels).

Reproduction NOAEL: at least 500 mg/kg.

Developmental NOAEL: 50 mg/kg (based on reduced post-natal growth starting at 150 mg/kg, accompanied by a decrease in serum T4 at 500 mg/kg).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 September 2016 to 29 December 2016

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:

Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016.

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650

Version / remarks:

The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Study Specific Test Item Information
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Not indicated

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies.
Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.
Source F0: Charles River Deutschland, Sulzfeld, Germany.
Age at start pretest: Females: approximately 10-12 weeks.
Age at start F0-treatment: Males: approximately 10-12 weeks. Females: approximately 12-14 weeks.
Number of F0-animals: 48 females and 40 males.
At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random and further used in the study. The remaining females were removed from the study.
Acclimatization F0: At least 5 days prior to start of pretest (females) or treatment (males).
Health inspection F0: At least upon receipt of the animals.
Randomization F0: Before initiation of pretest, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0: During pretest (females) and treatment (males and females): by earmark and tattoo.
Identification of pups: On PND 1, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

Animal Husbandry
Conditions: Environmental controls for the animal room were set to maintain 18 to 24 °C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod was between 07:00 and 19:00 hrs daily. The light/dark cycle was interrupted for study related activities. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation
Pre-test: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages
(MIII type, height 18 cm).
Lactation: Females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Lignocel S 8-15, JRS -J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 1.5 hours.
Water: Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 1.5 hours.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

Vehicle: Polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany).
Rationale for vehicle: Based on trial formulations performed at Charles River Den Bosch.
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.
Appearance of formulations: Solution (Groups 2-4).
Storage conditions: At room temperature.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Detection of mating was not confirmed for animal no. 74 which delivered live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Parturition: The females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Females that were littering were left undisturbed.
Number of pups: 418 pups.
Culling: To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (04 November 2016) according to a validated method (Test Facility Study No. 512475).
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-150 mg/mL) was determined as part of the analytical method development and validation study (Test Facility Study No. 512475).

Duration of treatment / exposure:

Males were dosed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were exposed for 50-56 days (most females) or 64 days (one female, no. 67), i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were exposed for 41 days.
Routinely, females that are littering are left undisturbed. In this study, female nos. 54 (Group 2) and 68 (Group 3) were not dosed on one occasion as they were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Details on study schedule:

No futher details specified.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

48 females and 40 males. (10 males/10 females per dose level).
At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random and further used in the study. The remaining females were removed from the study.

Control animals:

yes

Details on study design:

Based on the results of a dose range finding study in which dose limiting effects were noted at 1000 mg/kg, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 50, 150 and 500 mg/kg.

Positive control:

No required for this study.

Parental animals: Observations and examinations:

Mortality / Viability: At least twice daily.

Clinical signs: Clinical observations (detailed clinical signs and arena) were conducted from start of treatment onwards up to the day prior to necropsy at least at 1 hour (± 30 min)) after dosing based on the peak period of anticipated effects. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Functional Observations: The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
-hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
-fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
-locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable) and started at 1 hour (± 30 min) after dosing.

Body weights: Males and females were weighed on the first day of exposure (prior to first exposure) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum.

Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on
Days 0, 4, 7, 11, 14, 17 and 20 post-coitum.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Clinical Laboratory Investigations: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.

Haematology
The haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant, using the ADVIA® 2120i Hematology System (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands): Reported tabulated – see “Any other information” for parameters.
The clotting parameters were determined in plasma prepared with citrate as anticoagulant, using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France): Reported tabulated – see “Any other information” for parameters.

Clinical Biochemistry
The clinical biochemistry parameters were determined using the AU400 (Beckman Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in plasma, except for bile acids which were determined in serum: Reported tabulated – see “Any other information” for parameters.

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
During pretest, this was done for 48 females.
On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Not examined

Litter observations:

Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.
Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
Sex: Sex was determined for all pups on PND 1 and 4. Sex ratio (% male pups / % female pups) was calculated per group.
Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

Postmortem examinations (parental animals):

Termination
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
Necropsy was conducted on the following days:
Males: Following completion of the mating period (a minimum of 28 days of dose administration).
Females which delivered: PND 14-16.
Females which failed to deliver: Post-coitum Day 26 (females with evidence of mating)

Macroscopic Examination
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The number of former implantation sites were recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Selected 5 animals/sex/group:
Identification marks: not processed; Adrenal glands; (Aorta); Brain – cerebellum, mid-brain, cortex (7-levels); Caecum; Cervix; Clitoral gland; (Cowper’s gland); Duodenum; Epididymides; Eyes (with optic nerve (if detectable) and Harderian gland); Mammanry gland area (males and females); Femur including joint; (Glans penis); (Levator ani plus bulbocavernosus muscle complex (LABC)); Heart; Ileum; Jejunum; Kidneys; (Lacrimal gland, exorbital); (Larynx); Liver; Lung, infused with formalin; Lymph nodes – mandibular, mesenteric; (Nasopharynx); (Esophagus); Ovaries; (Pancreas); Payer’s patches [jejunum, ileum] if detectable; Pituitary gland; Preputial gland; Prostate gland; Rectum; (Salivary glands – mandibular, sublingual); Sciatic nerve; Seminal vesicles; Skeletal muscle; (Skin); Spinal cord –cervical, midthoratic, lumbar; Spleen; Sternum with bone marrow; Stomach; Testes; Thymus; Thyroid including parathyroid if detectable; (Tongue); Trachea; Urinary bladder; Uterus; Vagina; All gross lesions.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
All remaining animals:
Cervix; Clitoral gland; Coagulation gland; Cowper’s glands; Epididymides; Glans penis; Levator ani plus bulbocavernosus muscle complex (LABC); Mammary gland area (males and females); Ovaries; Preputial gland; Prostate gland; Seminal vesicles; Testes; Thyroid including parathyroid if detectable; Uterus; Vagina; All gross lesions; Identification marks: not processed.

Organ Weights
Terminal body weights were recorded from all rats at scheduled necropsy.
The following organ weights were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group:
Adrenal glands; Brain; Cowper’s glands; Epididymides; Glans penis; Heart; Kidneysl Levator ani plus bulbocavernosus muscle complex (LABC); Liver; Ovaries; Prostate; Seminal vesicles including coagulating glands; Spleen; Testes; Thymus; Thyroid; Uterus (including cervix)

All remaining animals:
Cowper’s glands; Epididymides; Glans penis; Levator ani plus bulbocavernosus muscle complex (LABC); Prostate; Seminal vesicles including coagulating glands; Testes; Thyroid (including parathyroid if detectable)
Absolute organ weights and organ to body weight ratios are reported.

Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

Histopathology
The following slides were examined by a pathologist:
-The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
-Additional slides of the testes of the selected 5 males of Groups 1 and 4, including the two males that failed to sire (nos. 01 and 31), to examine staging of spermatogenesis.
-All gross lesions of all animals (all dose groups).
-Spleen and liver of the selected 5 males and females of Groups 2 and 3 and thyroid gland of the selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
-The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Postmortem examinations (offspring):

F1-generation Pups - Pathology
F1-generation Pups - Termination
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 7-15), except those used for blood sampling on PND 13-15, were sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater, The Netherlands) by intraperitoneal (ip) injection.
Litter 45 pup no. 1 and litter 50 pup no. 11 (found dead during the weekend) were fixed in an identified container containing 70% ethanol (Klinipath, Duiven, The Netherlands) as they were not necropsied on the same day.
On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter were collected into one serum tube.
On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by exsanguination. Blood was collected into serum tubes.
Necropsy was conducted on the following days:
Condition Day of necropsy
Culling PND 4.
Terminal sacrifice PND 13-15.
Spontaneous deaths As soon as possible after death and always within 24 hours.

F1-generation pups - Macroscopic Examination
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test was applied to frequency data.
-The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:

Mating index (%)
(Number of females mated/Number of females paired) x 100

Precoital time
Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%)
(Number of pregnant females/Number of females paired) x 100

Conception index (%)
(Number of pregnant females/Number of females mated) x 100

Gestation index (%)
(Number of females bearing live pups/Number of pregnant females) x 100

Duration of gestation
Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

For each group, the following calculations were performed:

Post-implantation survival index (%)
(Total number of offspring born/Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%)
(Number of live offspring on Day 1 after littering/Total number of offspring born) x 100

Percentage live males at First Litter Check (%)
(Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100

Percentage of live females at First Litter Check (%)
(Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100

Viability index (%)
(Number of live offspring on Day 4 before culling/Number of live offspring on Day 1 after littering) x 100

Lactation index (%)
(Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 [after culling]) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during the daily clinical observations or during the weekly arena observations.
The clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was decreased in females at 150 and 500 mg/kg during the lactation period (statistically significant on Day 7 at 500 mg/kg and on Day 13 at both dose levels). At 500 mg/kg, this was accompanied by statistically significantly lower body weights on lactation Days 4-13. During the pre-mating and gestation periods, body weights and body weight gain of females were not affected by treatment.
Males showed no toxicologically relevant changes in body weight or body weight gain up to 500 mg/kg.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In females, food consumption before allowance for body weight was statistically significantly decreased at 150 and 500 mg/kg between Days 7-13 of lactation. Food consumption after allowance for body weight was similar between treated and control females throughout the lactation period.
In contrast, during gestation food consumption of treated females (before and after allowance for body weight) was higher compared to controls at 500 mg/kg (statistically significant at most time points) and, to a lesser extent, at 150 mg/kg (statistically significant on a few occasions).
During the pre-mating period, food consumption of females was not affected by treatment.
In males, food consumption before or after allowance for body weight was not affected by treatment up to 500 mg/kg.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant differences between treated animals and controls were noted in haematology parameters:
-Higher prothrombin time at 500 mg/kg in males.
-Higher total white blood cell count (WBC) at 500 mg/kg in males. This finding was likely to be unrelated to treatment as values at 500 mg/kg were well in the normal range, whereas the concurrent control group mean was at the lower end of this range.
-Lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) at 500 mg/kg in males, and lower mean corpuscular haemoglobin concentration (MCHC) at 500 mg/kg in females. These slight differences (values in treated rats remained within normal limits) were considered not to be toxicologically relevant as they occurred in the absence of treatment-related changes in haemoglobin, haematocrit or number of red blood cells.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
-Higher alanine aminotransferase (ALAT) at 500 mg/kg in both sexes (group means increased about twofold). Increased ALAT values were also observed in a few animals (one male, one female) at 150 mg/kg.
-Higher aspartate aminotransferase (ASAT) at 500 mg/kg in both sexes (group means increased about twofold). Increased ASAT values were also observed in a few animals (one male, two females) at 150 mg/kg and a single female at 50 mg/kg.
-Higher alkaline phosphatase (ALP) at 500 mg/kg in both sexes (group means increased about twofold). Increased ALP values were also observed in a few animals (one male, one female) at 150 mg/kg and two females at 50 mg/kg.
-Higher total bilirubin at 500 mg/kg in both sexes (group means increased about twofold).
-Higher bile acids at 500 mg/kg in males (group mean increased nearly fourfold).
-Lower total protein and albu min at 500 mg/kg in both sexes (group means decreased about 5-10%). Total protein was also lower (about 5%) at 150 mg/kg (not statistically significant in females).
-Lower fasting glucose at 150 and 500 mg/kg in females (group means decreased about 15 and 30%, respectively).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the liver at 150 and 500 mg/kg, and in the spleen at 500 mg/kg. These changes occurred in both sexes.
In the liver, single cell necrosis (mainly centrilobular) and increased mitosis was present in males and females starting from 150 mg/kg up to moderate and slight degree, respectively.
Pigmentation of macrophages and/or Kupffer cells was observed in males at 500 mg/kg at minimal degree and in females starting from 150 mg/kg up to slight degree. Additionally, in females hypertrophy/hyperplasia of Kupffer cells was observed starting from 150 mg/kg up to slight degree.
In the spleen, a decreased incidence of extramedullary hematopoiesis was present in males and females at 500 mg/kg, and an increased severity of pigmentation was present in females at 500 mg/kg (minimal to slight pigmentation is considered to be normal background).
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone analyses:
Serum levels of T4 in F0 males were statistically significantly lower at 500 mg/kg (relative difference from controls: 22%).
The statistically significantly higher T4 level in males at 50 mg/kg was considered to be unrelated to treatment due to the absence of similar changes at the higher dose levels.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment.
Most females had regular cycles of 4 days. Extended di-estrus occurred in female no. 67 at 150 mg/kg, and irregular cycles occurred in two other females at 150 mg/kg (nos. 63 and 65). Females no. 67 and 65 had normal litters. No. 63 delivered only five pups (two of these were found dead at first litter check and one went missing on PND 2). These findings were considered to be unrelated to treatment because their incidence showed no dose-related trend.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

One control female (no. 41, mated with male no. 1) and one female treated at 500 mg/kg (no. 71, mated with male no. 31) were not pregnant despite proof of mating. Histopathology did not reveal any changes in the reproductive organs that could explain this.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and spermatogenic staging profiles were normal for all males examined.

Body weight gain was decreased, to about the same extent, in females at 150 and 500 mg/kg throughout the lactation period. This was accompanied by decreased body weights at 500 mg/kg (up to 10% lower than controls at Day 13 of lactation) and decreased food consumption between lactation Days 7-13 at 150 and 500 mg/kg. Food consumption during gestation, on the other hand, was increased in females at 500 mg/kg and, to a lesser extent, 150 mg/kg. Body weight (gain) and food consumption of treated males were not adversely affected.

No treatment-related changes were noted in the remaining parental in-life parameters investigated in this study (i.e. mortality, clinical appearance and functional observations).

Microscopic examination revealed treatment-related changes in the liver of males and females treated at 150 or 500 mg/kg, characterized by single cell necrosis, increased mitosis, pigmentation of Kupffer cells/macrophages and hypertrophy/hyperplasia of Kupffer cells.
Based on the primary degenerative nature of the liver findings (in particular single cell necrosis), these findings were considered to be adverse. The increased mitosis was most likely a compensatory response to the single cell necrosis and also the pigmentation of Kupffer cells/macrophages and hypertrophy/hyperplasia of the Kupffer cells were considered to be secondary to this degenerative process. The somewhat lower weight of the liver in females at 500 mg/kg could not clearly be related to the microscopic liver findings. Liver weight of treated males was not affected.

At 500 mg/kg (in both sexes), the histopathological changes in the liver were accompanied by increases in the plasma levels of alanine and aspartate aminotransferase (ALAT, ASAT), alkaline phosphatase (ALP), bilirubin and bile acids, and decreases in total protein and albumin. To a lesser extent, a decrease in total protein and increases in plasma enzymes were also observed 150 mg/kg (enzymes were increased in a few animals). Additionally, fasting glucose was decreased in females at 150 and 500 mg/kg.
At 50 mg/kg, ASAT and ALP were increased in one or two females. As these clinical pathology findings at 50 mg/kg occurred in the absence of histopathological evidence of hepatotoxicity, they were considered to be non-adverse.

At microscopic examination of the spleen, treatment-related changes were observed at 500 mg/kg. These changes consisted of a decreased incidence of extramedullary hematopoiesis in both sexes and a slightly increased severity of pigmentation in females.
These findings were considered to be non-adverse since haematology parameters were not adversely affected by treatment.

Males treated at 500 mg/kg had lower weights of the prostate gland, Cowper’s gland and ani plus bulbocavernosus muscle complex (LABC). These treatment-related organ weight changes occurred in the absence of macroscopic or microscopic correlate and were therefore considered not to be adverse. Females at 500 mg/kg had lower thymus weights, which correlated with a reduced size of the thymus in one animal. In the absence of a microscopic correlate, the decrease in thymus weight was regarded as non-adverse.

The serum level of thyroid hormone T4 was decreased in males at 500 mg/kg (T4 in females was not measured). T4 values in treated males remained within normal limits, and there were no accompanying changes in the weight or morphology of the thyroid. As such, the decrease in T4 was considered not to represent an adverse change.

Haematology showed a prolongation of prothrombin time (PT) in males at 500 mg/kg. This was not accompanied by a relevant change in activated partial thromboplastin time (APTT) and there was no evidence of impaired coagulation. Possibly, the prolongation of PT was associated with the adverse effect on the liver (decreased hepatic synthesis of clotting factors).

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg).

No treatment-related or toxicologically relevant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
The incidental clinical signs observed remained within the range considered normal for pups of this age and showed no dose-related trend. They were therefore considered to be unrelated o treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices across the groups were 98 or 100%.
At first litter check, two pups of the control group (litter nos. 45 and 50) and two pups at 150 mg/kg (litter no. 63) were found dead. This incidental pup mortality was unrelated to treatment with the test item.
The mean number of living pups at first litter check was slightly lower at 150 and 500 mg/kg.
The differences from the concurrent control group were not statistically significant and could partly be explained by the small size of a single litter in these groups (litter nos. 63 and 78, each consisting of three pups). Such a small litter size occasionally occurs in untreated controls. The small size of litter no. 78 correlated with a low number of implantation sites in the dam. All other litters at 150 and 500 mg/kg had normal sizes (between 8 and 14 pups).
For these reasons, the variations in litter size were considered not to be toxicologically relevant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At PND 13, body weights of male and female pups were decreased at 150 and 500 mg/kg.
The relative differences from controls were generally statistically significant and amounted to about 10% at 150 mg/kg and 15% at 500 mg/kg.
Body weights of pups at birth (PND 1) were similar across the groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No pups were found dead or went missing between lactation Days 5 and 13. For each group, the number of live offspring on Day 13 after littering was the same as the number of live offspring on Day 4 (after culling), resulting in a lactation index of 100% for all groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups were statistically significantly decreased at 500 mg/kg (relative difference from controls: 29% in males, 25% in females).

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment. Findings were limited to absence of milk in the stomach or cannibalism in the few pups found dead at first litter check.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

At PND 13, body weights of male and female pups were decreased at 150 and 500 mg/kg (by about 10 and 15%, respectively). Pup body weights at PND 1 were similar across the groups, indicating that the lower weights at PND 13 resulted from reduced post-natal growth.
The serum level of thyroid hormone T4 was decreased in male and female pups at 500 mg/kg (on average by about 25-30%).
These effects on post-natal growth and T4 were considered to be toxicologically relevant. They occurred in the presence of maternal toxicity. No developmental toxicity was observed in the absence of maternal toxicity.
No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and the early postnatal pup developmental parameters mortality, clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males) and macroscopy.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Reproductive effects observed:

no

CLINICAL SIGNS SUMMARY

MALES

		PRE MATING														REPRO PERIOD
SIGN (MAX. GRADE)	WEEK:	1	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.	.	.	.	.	.	.	.	.	.	.	.	.
(LOCATION)	DAY:	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7
GROUP 1 (CONTROL) Secretion / excretion  Salivation (3)	G: %:	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	1 1	. .	. .	. .	. .	. .	. .
GROUP 2 (50 MG/KG) Skin /fur  Scabs (3) (Tail)	G: %:	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	1 1	1 1	1 1	1 1	1 1	1 1	. .	. .
GROUP 3 (150 MG/KG) No clinical signs noted
GROUP 4 (500 MG/KG) Secretion / excretion  Salivation (3)	G: %:	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	1 1

 

CLINICAL SIGNS SUMMARY

FEMALES

		PRE MATING														REPRO PERIOD
SIGN (MAX. GRADE)	WEEK:	1	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.	.	.	.	.	.	.	.	.	.	.	.	.
(LOCATION)	DAY:	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7
GROUP 1 (CONTROL) No clinical signs noted
GROUP 2 (50 MG/KG) No clinical signs noted
GROUP 3 (150 MG/KG) No clinical signs noted
GROUP 4 (500 MG/KG) No clinical signs noted

G: Median value of the highest individual daily grades.

%: Percent of affected animal (0=less than 5%, 1=between 5% and 15%,….,A=more than 95%)

.: Observation performed, sign no present

BODY WEIGHTS (GRAM) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
PRE MATING
DAY 1 WEEK 1	MEAN ST. DEV N	320 11.5 10	318 8.3 10	314 14.4 10	320 12.3 10
DAY 8 WEEK 2	MEAN ST. DEV N	340 16.1 10	337 12.3 10	337 15.7 10	344 15.5 10
MATING PERIOD
DAY 1 WEEK 1	MEAN ST. DEV N	358 21.1 10	351 18.8 10	350 17.6 10	347 17.1 10
DAY 8 WEEK 2	MEAN ST. DEV N	368 23.0 10	365 21.7 10	362 19.7 10	354 15.5 10
DAY 15 WEEK 3	MEAN ST. DEV N	378 24.2 10	376 35.6 10	373 20.0 10	366 16.1 10

FEMALES

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
PRE MATING
DAY 1 WEEK 1	MEAN ST. DEV N	212 11.4 10	218 10.1 10	216 10.4 10	219 7.7 10
DAY 8 WEEK 2	MEAN ST. DEV N	218 8.3 10	225 13.2 10	223 11.0 10	222 8.9 10
MATING PERIOD
DAY 1 WEEK 1	MEAN ST. DEV N	225 9.9 10	230 13.4 10	228 13.8 10	226 12.0 10
DAY 8 WEEK 2	MEAN ST. DEV N			232 --- 1	222 --- 1
DAY 15 WEEK 3	MEAN ST. DEV N				245 --- 1
DAY 22 WEEK 4	MEAN ST. DEV N				283 --- 1

 

HAEMATOLOGY SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT
WBC 10E9/L	MEAN ST. DEV N	5.8 1.8 4	6.4 1.1 5	8.1 1.7 5	8.6* 1.7 5
Neutrophils %WBC	MEAN ST. DEV N	27.0 19.3 4	15.6 5.7 5	13.1 4.1 5	13.3 2.5 5
Lymphocytes %WBC	MEAN ST. DEV N	70.3 20.1 4	81.8 5.8 5	83.9 4.6 5	84.4 2.7 5
Monocytes %WBC	MEAN ST. DEV N	1.4 0.6 4	1.7 0.4 5	2.1 0.9 5	1.6 0.4 5
Eosinophils %WBC	MEAN ST. DEV N	1.2 0.6 4	0.8 0.2 5	0.8 0.3 5	0.6 0.1 5
Basophils %WBC	MEAN ST. DEV N	0.1 0.1 4	0.1 0.1 5	0.1 0.0 5	0.1 0.1 5
Red blood cells 10412/L	MEAN ST. DEV N	8.83 0.22 4	8.90 0.12 5	8.99 0.31 5	9.21 0.32 5
Reticulocytes %RBC	MEAN ST. DEV N	2.5 0.5 4	2.8 0.6 5	2.7 0.4 5	3.0 0.4 5
RDW %	MEAN ST. DEV N	12.5 0.4 4	12.4 0.7 5	11.8 0.4 5	12.4 0.8 5
Haemoglobin mmol/L	MEAN ST. DEV N	9.8 0.4 4	9.6 0.3 5	9.6 0.2 5	9.7 0.1 5
Haematocrit L/L	MEAN ST. DEV N	0.463 0.015 4	0.457 0.007 5	0.467 0.011 5	0.469 0.015 5
MCV fL	MEAN ST. DEV N	52.4 0.6 4	51.3 0.7 5	52.0 1.0 5	50.9* 0.9 5
MCH fmol	MEAN ST. DEV N	1.11 0.04 4	1.08 0.02 5	1.07 0.02 5	1.06* 0.03 5
MCHC mmol/L	MEAN ST. DEV N	21.15 0.60 4	21.07 0.52 5	20.57 0.25 5	20.80 0.45 5
Platelets 10E9/L	MEAN ST. DEV N	769 69 4	730 59 5	782 88 5	835 122 5
PT s	MEAN ST. DEV N	17.1 1.0 4	17.0 0.3 5	17.9 0.4 5	18.5** 0.3 4
APTT s	MEAN ST. DEV N	14.0 1.6 4	15.0 1.7 5	15.4 2.2 5	13.2 0.5 4

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

HAEMATOLOGY SUMMARY

FEMALES

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT
WBC 10E9/L	MEAN ST. DEV N	7.1 1.4 5	7.4 2.4 5	6.0 1.3 5	8.7 2.1 5
Neutrophils %WBC	MEAN ST. DEV N	41.4 11.5 5	41.4 3.9 5	48.2 19.7 5	46.2 8.3 5
Lymphocytes %WBC	MEAN ST. DEV N	53.8 10.7 5	54.0 3.6 5	46.6 20.6 5	49.2 7.9 5
Monocytes %WBC	MEAN ST. DEV N	3.6 1.8 5	3.5 0.7 5	4.2 1.4 5	3.7 1.6 5
Eosinophils %WBC	MEAN ST. DEV N	0.9 0.5 5	0.9 0.4 5	1.0 0.3 5	0.8 0.1 5
Basophils %WBC	MEAN ST. DEV N	0.1 0.1 5	0.1 0.1 5	0.1 0.0 5	0.1 0.0 5
Red blood cells 10412/L	MEAN ST. DEV N	7.73 0.71 5	7.65 0.43 5	8.08 0.29 5	7.85 0.27 5
Reticulocytes %RBC	MEAN ST. DEV N	3.7 0.9 5	4.1 0.6 5	3.7 0.3 5	3.3 0.7 5
RDW %	MEAN ST. DEV N	14.2 0.4 5	14.7 1.9 5	13.3 1.1 5	14.1 2.3 5
Haemoglobin mmol/L	MEAN ST. DEV N	9.5 0.9 5	9.4 0.1 5	9.7 0.4 5	9.3 0.5 5
Haematocrit L/L	MEAN ST. DEV N	0.443 0.046 5	0.447 0.013 5	0.463 0.021 5	0.448 0.021 5
MCV fL	MEAN ST. DEV N	57.3 1.5 5	58.5 1.8 5	57.3 1.4 5	57.1 2.3 5
MCH fmol	MEAN ST. DEV N	1.23 0.04 5	1.24 0.06 5	1.20 0.03 5	1.19 0.06 5
MCHC mmol/L	MEAN ST. DEV N	21.40 0.51 5	21.12 0.37 5	21.00 0.19 5	20.77* 0.29 5
Platelets 10E9/L	MEAN ST. DEV N	840 136 5	941 79 5	850 68 5	842 175 5
PT s	MEAN ST. DEV N	17.0 0.8 4	16.9 0.7 5	16.6 0.4 5	17.4 0.6 5
APTT s	MEAN ST. DEV N	13.0 1.7 4	12.9 2.0 5	13.3 2.4 5	14.1 1.0 5

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

CLINICAL BIOCHEMISTRY SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT
ALAT U/L	MEAN ST. DEV N	40.4 2.1 5	53.0 22.4 5	52.8 17.6 5	98.8** 11.0 5
ASAT U/L	MEAN ST. DEV N	77.2 3.7 5	92.5 31.1 5	99.5 18.8 5	152.4** 35.2 5
ALP U/L	MEAN ST. DEV N	136 34 5	162 27 5	180 22 5	285** 73 5
Total protein g/L	MEAN ST. DEV N	64.2 2.6 5	62.1 1.3 5	60.8 1.8 5	57.3** 1.9 5
Albumin g/L	MEAN ST. DEV N	33.9 1.2 5	62.1 1.3 5	60.8* 1.8 5	57.3* 1.9 5
Total bilirubin umol/L	MEAN ST. DEV N	2.3 0.3 5	2.3 0.3 5	3.1 0.4 5	5.8** 1.2 5
Urea mmol/L	MEAN ST. DEV N	6.9 1.6 5	5.8 0.9 5	7.5 1.1 5	8.6 1.4 5
Creatinine umol/L	MEAN ST. DEV N	37.5 2.3 5	36.9 1.9 5	38.6 1.6 5	35.8 1.2 5
Glucose mmol/L	MEAN ST. DEV N	8.71 1.36 5	9.61 0.80 5	9.02 0.66 5	7.81 0.36 5
Cholesterol mmol/L	MEAN ST. DEV N	2.39 0.52 5	1.81 0.28 5	1.96 0.21 5	2.55 0.51 5
Bile Acids umol/L	MEAN ST. DEV N	34.9 17.2 5	23.2 15.0 5	30.2 13.0 5	128.8* 93.5 5
Sodium mmol/L	MEAN ST. DEV N	140.8 0.6 5	140.6 0.3 5	140.5 0.6 5	140.0 1.3 5
Potassium mmol/L	MEAN ST. DEV N	4.12 0.25 5	4.00 0.06 5	3.94 0.16 5	4.09 0.16 5
Chloride mmol/L	MEAN ST. DEV N	103 1 5	103 0 5	103 1 5	103 2 5
Calcium mmol/L	MEAN ST. DEV N	2.59 0.10 5	2.53 0.02 5	2.52 0.05 5	2.52 0.03 5
Inorg.Phos mmol/L	MEAN ST. DEV N	1.97 0.09 5	1.86 0.10 5	1.88 0.15 5	2.12 0.15 5
Total T4 ug/dL	MEAN ST. DEV N	4.09 0.60 10	5.13* 0.69 10	4.71 1.12 10	3.20* 0.52 10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

CLINICAL BIOCHEMISTRY SUMMARY

FEMALES

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT
ALAT U/L	MEAN ST. DEV N	71.5 9.0 5	67.9 23.8 5	99.1 40.2 5	144.5* 71.4 5
ASAT U/L	MEAN ST. DEV N	114.5 20.0 5	132.4 62.3 5	175.3 66.1 5	216.6* 65.4 5
ALP U/L	MEAN ST. DEV N	119 36 5	178 76 5	171 48 5	265* 127 5
Total protein g/L	MEAN ST. DEV N	64.1 3.4 5	63.4 1.5 5	60.1 3.9 5	57.0** 2.1 5
Albumin g/L	MEAN ST. DEV N	32.7 1.3 5	32.6 1.0 5	31.1 1.4 5	29.5** 0.8 5
Total bilirubin umol/L	MEAN ST. DEV N	2.4 0.7 5	2.7 0.5 5	2.8 0.3 5	4.5** 1.4 5
Urea mmol/L	MEAN ST. DEV N	10.3 1.5 5	10.2 1.1 5	11.1 2.1 5	12.4 2.1 5
Creatinine umol/L	MEAN ST. DEV N	43.3 2.5 5	44.2 2.1 5	45.1 4.4 5	45.0 4.4 5
Glucose mmol/L	MEAN ST. DEV N	9.12 0.76 5	8.11 0.86 5	45.1 4.4 5	45.0 4.4 5
Cholesterol mmol/L	MEAN ST. DEV N	2.41 0.34 5	2.66 0.35 5	3.10 0.60 5	3.02 0.82 5
Bile Acids umol/L	MEAN ST. DEV N	38.6 35.7 5	50.1 7.9 5	47.2 24.0 5	120.6 165.0 5
Sodium mmol/L	MEAN ST. DEV N	135.4 1.4 5	137.3 2.0 5	135.7 2.4 5	135.7 1.6 5
Potassium mmol/L	MEAN ST. DEV N	4.04 0.28 5	3.91 0.20 5	4.22 0.19 5	4.35 0.22 5
Chloride mmol/L	MEAN ST. DEV N	98 2 5	98 2 5	99 2 5	100 3 5
Calcium mmol/L	MEAN ST. DEV N	2.74 0.09 5	2.70 0.13 5	2.67 0.06 5	2.65 0.11 5
Inorg.Phos mmol/L	MEAN ST. DEV N	2.64 0.47 5	2.84 0.44 5	2.80 0.52 5	2.84 0.49 5
Total T4 ug/dL	MEAN ST. DEV N	--- --- 0	--- --- 0	--- --- 0	--- --- 0

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

MACROSCOPIC FINDINGS SUMMARY

MALES

	GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT
Animals examined	10	10	10	10
Animals without findings	8	8	8	9
Animals affected	2	2	2	1
Liver  Diaphragmatic hernia	0	1	0	0
Kidneys  Pelvic dilation	0	1	0	0
Epididymides  Nodule(s)	0	1	0	0
Cowper’s gland  Enlarged	0	1	0	0
Thyroid gland  Discolouration  Agenesis	0 1	0 0	1 0	0 0
Thymus  Focus/foci  Discolouration	0 1	0 0	0 0	1 0
Skin  Nodule(s)	0	0	1	0

FEMALES

	GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT
Animals examined	10	10	10	10
Animals without findings	7	10	8	6
Animals affected	3	0	2	4
Stomach  Focus/foci	1	0	1	0
Jejunum  Discolouration	0	0	1	0
Uterus  Contains fluid	0	0	0	1
Clitoral glands  Nodule(s)  Focus/foci	0 0	0 0	0 1	1 0
Thymus  Reduced in size  Discolouration	0 0	0 0	0 1	1 0
Mandibular lymph n  Discolouration	2	0	0	0
Skin  Scab formation	0	0	0	1

#/## Fischer’s Exact test significant at 5% (#) or 1% (##) level

ORGAN WEIGHTS (GRAM) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT
BODY W. (GRAM)	MEAN ST. DEV N	357 21 10	354 23 10	344 22 10	335 15 10
BRAIN (GRAM)	MEAN ST. DEV N	2.04 0.07 5	2.02 0.05 5	2.02 0.03 5	2.06 0.05 5
HEART (GRAM)	MEAN ST. DEV N	0.955 0.065 5	0.922 0.072 5	0.894 0.059 5	0.962 0.135 5
LIVER (GRAM)	MEAN ST. DEV N	8.77 1.06 5	8.66 1.10 5	7.76 0.98 5	7.56 0.79 5
THYROIDS (GRAM)	MEAN ST. DEV N	0.014 0.004 10	0.016 0.004 10	0.017 0.005 10	0.015 0.003 10
THYMUS (GRAM)	MEAN ST. DEV N	0.330 0.065 5	0.346 0.102 5	0.349 0.107 5	0.320 0.031 5
KIDNEYS (GRAM)	MEAN ST. DEV N	2.52 0.31 5	2,51 0.15 5	2.46 0.11 5	2.68 0.20 5
ADRENALS (GRAM)	MEAN ST. DEV N	0.063 0.010 5	0.070 0.011 5	0.054 0.008 5	0.052 0.004 5
SPLEEN (GRAM)	MEAN ST. DEV N	0.583 0.133 5	0.554 0.034 5	0.602 0.072 5	0.587 0.047 5
TESTES (GRAM)	MEAN ST. DEV N	3.58 0.18 10	3.57 0.24 10	3.55 0.20 10	3.62 0.31 10
GLANS PENIS (GRAM)	MEAN ST. DEV N	0.126 0.014 10	0.127 0.016 10	0.127 0.019 10	0.119 0.019 10
PROSTATE ! (GRAM)	MEAN ST. DEV N	1.089 0.186 5	1.029 0.157 5	1.084 0.212 5	0.843 0.204 5
LABC (GRAM)	MEAN ST. DEV N	1.136 0.100 10	1.149 0.138 10	1.095 0.106 10	0.940** 0.116 10
PROSTATE GLAND (GRAM)	MEAN ST. DEV N	0.923 0.139 5	0.945 0.090 5	0.873 0.130 5	0.747** 0.125 5
COWPER’S GLANDS (GRAM)	MEAN ST. DEV N	0.107 0.014 10	0.101 0.019 10	0.097 0.014 10	0.079** 0.016 10
EPIDIDYMIDES (GRAM)	MEAN ST. DEV N	1.145 0.075 10	1.171 0.053 10	1.115 0.074 10	1.094 0.060 10
SEMINAL VES ! (GRAM)	MEAN ST. DEV N	1.655 0.150 5	1.575 0.092 5	1.277* 0.257 5	1.051* 0.174 5
SEMINAL VESICLES (GRAM)	MEAN ST. DEV N	1.382 0.213 5	1.465 0.252 5	1.204 0.160 5	1.131 0.112 5

! Organ weight after fixation

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

ORGAN/BODY WEIGHT RATIOS (%) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT
BODY W. (GRAM)	MEAN ST. DEV N	357 21 10	354 23 10	344 22 10	335 15 10
BRAIN (%)	MEAN ST. DEV N	0.56 0.02 5	0.58 0.03 5	0.59 0.03 5	0.61 0.04 5
HEART (%)	MEAN ST. DEV N	0.264 0.08 5	0.265 0.020 5	0.262 0.022 5	0.284 0.026 5
LIVER (%)	MEAN ST. DEV N	2.42 0.19 5	2.48 0.19 5	2.26 0.15 5	2.24 0.14 5
THYROIDS (%)	MEAN ST. DEV N	0.004 0.001 10	0.005 0.001 10	0.005 0.001 10	0.005 0.001 10
THYMUS (%)	MEAN ST. DEV N	0.091 0.016 5	0.099 0.028 5	0.102 0.030 5	0.095 0.008 5
KIDNEYS (%)	MEAN ST. DEV N	0.69 0.06 5	0.72 0.04 5	0.72 0.02 5	0.79** 0.03 5
ADRENALS (%)	MEAN ST. DEV N	0.017 0.002 5	0.020 0.002 5	0.016 0.002 5	0.015 0.001 5
SPLEEN (%)	MEAN ST. DEV N	0.160 0.029 5	0.159 0.008 5	0.176 0.018 5	0.174 0.009 5
TESTES (%)	MEAN ST. DEV N	1.01 0.09 10	1.01 0.07 10	1.04 0.10 10	1.08 0.11 10
GLANS PENIS (%)	MEAN ST. DEV N	0.035 0.003 10	0.036 0.007 10	0.037 0.005 10	0.035 0.005 10
PROSTATE ! (%)	MEAN ST. DEV N	0.310 0.048 5	0.287 0.050 5	0.312 0.045 5	0.354 0.063 5
LABC (%)	MEAN ST. DEV N	0.318 0.022 10	0.326 0.047 10	0.319 0.027 10	0.280 0.028 10
PROSTATE GLAND (%)	MEAN ST. DEV N	0.255 0.035 5	0.273 0.036 5	0.257 0.049 5	0.221 0.035 5
COWPER’S GLANDS (%)	MEAN ST. DEV N	0.030 0.004 10	0.029 0.007 10	0.028 0.003 10	0.023* 0.004 10
EPIDIDYMIDES (%)	MEAN ST. DEV N	0.322 0.025 10	0.332 0.030 10	0.325 0.018 10	0.327 0.019 10
SEMINAL VES ! (%)	MEAN ST. DEV N	0.474 0.065 5	0.438 0.038 5	0.368* 0.054 5	0.315** 0.045 5
SEMINAL VESICLES (%)	MEAN ST. DEV N	0.384 0.074 5	0.424 0.086 5	0.352 0.046 5	0.336 0.032 5

! Organ weight after fixation

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

ORGAN WEIGHTS (GRAM) SUMMARY

FEMALES

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT
BODY W. (GRAM)	MEAN ST. DEV N	255 13 10	264 11 10	248 12 10	238* 16 9
BRAIN (GRAM)	MEAN ST. DEV N	1.87 0.08 5	1.85 0.06 5	1.91 0.07 5	1.83 0.03 5
HEART (GRAM)	MEAN ST. DEV N	0.817 0.020 5	0.810 0.072 5	0.785 0.063 5	0.765 0.032 5
LIVER (GRAM)	MEAN ST. DEV N	9.18 0.55 5	9.34 1.01 5	8.71 0.59 5	7.56** 0.56 5
THYROIDS (GRAM)	MEAN ST. DEV N	0.014 0.003 10	0.013 0.002 10	0.014 0.003 10	0.013 0.002 10
THYMUS (GRAM)	MEAN ST. DEV N	0.263 0.052 5	0.260 0.049 5	0.210 0.042 5	0.160* 0.055 5
KIDNEYS (GRAM)	MEAN ST. DEV N	1.99 0.17 5	1.95 0.09 5	2.02 0.10 5	1.87 0.12 5
ADRENALS (GRAM)	MEAN ST. DEV N	0.081 0.010 5	0.071 0.010 5	0.067 0.008 5	0.060* 0.013 5
SPLEEN (GRAM)	MEAN ST. DEV N	0.562 0.081 5	0.570 0.076 5	0.611 0.298 5	0.424 0.058 5
OVARIES (GRAM)	MEAN ST. DEV N	0.127 0.021 5	0.132 0.004 5	0.112 0.016 5	0.109 0.012 5
UTERUS (GRAM)	MEAN ST. DEV N	0.525 0.022 5	0.514 0.060 5	0.459 0.081 5	0.406** 0.040 5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

ORGAN/BODY WEIGHT RATIOS (%) SUMMARY

FEMALES

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT
BODY W. (GRAM)	MEAN ST. DEV N	255 13 10	264 11 10	248 12 10	238* 16 9
BRAIN (%)	MEAN ST. DEV N	0.71 0.02 5	0.69 0.03 5	0.76 0.03 5	0.75 0.04 5
HEART (%)	MEAN ST. DEV N	0.312 0.015 5	0.303 0.017 5	0.309 0.017 5	0.311 0.018 5
LIVER (%)	MEAN ST. DEV N	3.50 0.19 5	3.49 0.26 5	3.42 0.21 5	3.11* 0.26 5
THYROIDS (%)	MEAN ST. DEV N	0.005 0.001 10	0.005 0.001 10	0.006 0.001 10	0.005 0.001 9
THYMUS (%)	MEAN ST. DEV N	0.100 0.019 5	0.097 0.017 5	0.079 0.015 5	0.066* 0.020 5
KIDNEYS (%)	MEAN ST. DEV N	0.76 0.05 5	0.73 0.05 5	0.79 0.04 5	0.77 0.04 5
ADRENALS (%)	MEAN ST. DEV N	0.031 0.003 5	0.027 0.003 5	0.026 0.003 5	0.025 0.006 5
SPLEEN (%)	MEAN ST. DEV N	-.214 0.026 5	0.213 0.026 5	0.242 0.120 5	0.174 0.027 5
OVARIES (%)	MEAN ST. DEV N	0.048 0.006 5	0.050 0.002 5	0.044 0.006 5	0.045 0.004 5
UTERUS (%)	MEAN ST. DEV N	0.201 0.015 5	0.192 0.017 5	0.181 0.034 5	0.167 0.020 5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Conclusions:

Based on the results, the following No Observed Adverse Effect Levels (NOAELs) were derived:
Parental NOAEL: 50 mg/kg (based on adverse microscopic alterations in the liver and associated changes in clinical pathology parameters in both sexes, and reduced body weight gain and food consumption during lactation in females at the higher dose levels).
Reproduction NOAEL: at least 500 mg/kg.
Developmental NOAEL: 50 mg/kg (based on reduced post-natal growth starting at 150 mg/kg, accompanied by a decrease in serum T4 at 500 mg/kg).

Executive summary:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of formaldehyde, oligomeric reaction products with acetone and diphenylamine in rats by oral gavage.

 

The study was based on the following guidelines:

-OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016.

-OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

-OECD 421, Reproduction/Developmental Toxicity Screening Test, July 2016.

-OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

-EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

-OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

-OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

 

Based on the results of a dose range finding study in which dose limiting effects were noted at 1000 mg/kg, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 50, 150 and 500 mg/kg.

 

The test item, formulated in polyethylene glycol 400, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

 

Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated for 50-56 days (most females) or 64 days (one female), i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 13 days of lactation. Females which failed to deliver offspring were treated for 41 days.

 

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy).

Formulations were analyzed once during the study to assess accuracy and homogeneity.

 

Accuracy and homogeneity of formulations were demonstrated by analyses.

Parental results:

Body weight gain was decreased, to about the same extent, in females at 150 and 500 mg/kg throughout the lactation period. This was accompanied by decreased body weights at 500 mg/kg (up to 10% lower than controls at Day 13 of lactation) and decreased food consumption between lactation Days 7-13 at 150 and 500 mg/kg. Food consumption during gestation, on the other hand, was increased in females at 500 mg/kg and, to a lesser extent, 150 mg/kg.

 

Adverse, treatment-related microscopic changes were observed in the liver starting at 150 mg/kg, generally in both sexes. These changes were characterized by single cell necrosis (up to moderate degree), increased mitosis (up to slight degree), pigmentation of Kupffer cells/macrophages (up to slight degree; not observed in 150 mg/kg males) and hypertrophy/hyperplasia of Kupffer cells (females only). Liver weight was slightly decreased in females at 500 mg/kg but this could not clearly be related to the microscopic liver findings.

 

At 500 mg/kg (in both sexes), the histopathological changes in the liver were accompanied by increases in the plasma levels of alanine and aspartate aminotransferase (ALAT, ASAT), alkaline phosphatase (ALP), bilirubin and bile acids, and decreases in total protein and albumin. To a lesser extent, a decrease in total protein and increases in plasma enzymes were also observed 150 mg/kg (enzymes were increased in a few animals). Additionally, fasting glucose was decreased in females at 150 and 500 mg/kg. The increased ASAT and ALP values in a few females at 50 mg/kg were not corroborated by histopathological changes and therefore considered to be non-adverse.

 

Microscopic examination of the spleen showed a treatment-related decrease in the incidence of extramedullary hematopoiesis in both sexes and a slightly increased severity of pigmentation in females at 500 mg/kg. In the absence of adverse changes in haematology parameters, these findings were considered to be non-adverse.

 

Males treated at 500 mg/kg had lower weights of the prostate gland, Cowper’s gland and ani plus bulbocavernosus muscle complex (LABC). These treatment-related organ weight changes occurred in the absence of macroscopic or microscopic correlate and were therefore considered not to be adverse. Females at 500 mg/kg had lower thymus weights, which correlated with a reduced size of the thymus in one animal. In the absence of a microscopic correlate, the decrease in thymus weight was regarded as non-adverse.

 

The serum level of thyroid hormone T4 was decreased by about 20% in males at 500 mg/kg.

The weight or morphology of the thyroid were not affected by treatment. Therefore, the decrease in T4 was regarded as non-adverse.

 

Prothrombin time (PT) was increased in males at 500 mg/kg. There was no evidence of impaired coagulation. Possibly, the prolonged PT was associated with the adverse effect on the liver (decreased hepatic synthesis of clotting factors).

 

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg).

 

Developmental results:

At PND 13, body weights of male and female pups were decreased at 150 and 500 mg/kg (by about 10 and 15%, respectively). Pup body weights at PND 1 were similar across the groups, indicating that the lower weights at PND 13 resulted from reduced post-natal growth.

 

The serum level of thyroid hormone T4 was decreased in male and female pups at 500 mg/kg (on average by about 25-30%).

 

These developmental effects were considered to be toxicologically relevant. They occurred in the presence of maternal toxicity. No developmental toxicity was observed in the absence of maternal toxicity.

 

Conclusion

Based on these results, the following No Observed Adverse Effect Levels (NOAELs) were derived:

Parental NOAEL: 50 mg/kg (based on adverse microscopic alterations in the liver and associated changes in clinical pathology parameters in both sexes, and reduced body weight gain and food consumption during lactation in females at the higher dose levels).

Reproduction NOAEL: at least 500 mg/kg.

Developmental NOAEL: 50 mg/kg (based on reduced post-natal growth starting at 150 mg/kg, accompanied by a decrease in serum T4 at 500 mg/kg).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with formaldehyde, oligomeric reaction products with acetone and diphenylamine in rats by oral gavage.

 

The test item, formulated in polyethylene glycol 400, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated for 50-56 days (most females) or 64 days (one female), i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 13 days of lactation. Females which failed to deliver offspring were treated for 41 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy).

Parental results:

Body weight gain was decreased, to about the same extent, in females at 150 and 500 mg/kg throughout the lactation period. This was accompanied by decreased body weights at 500 mg/kg (up to 10% lower than controls at Day 13 of lactation) and decreased food consumption between lactation Days 7-13 at 150 and 500 mg/kg. Food consumption during gestation, on the other hand, was increased in females at 500 mg/kg and, to a lesser extent, 150 mg/kg.

Adverse, treatment-related microscopic changes were observed in the liver starting at 150 mg/kg, generally in both sexes. These changes were characterized by single cell necrosis (up to moderate degree), increased mitosis (up to slight degree), pigmentation of Kupffer cells/macrophages (up to slight degree; not observed in 150 mg/kg males) and hypertrophy/hyperplasia of Kupffer cells (females only). Liver weight was slightly decreased in females at 500 mg/kg but this could not clearly be related to the microscopic liver findings. At 500 mg/kg (in both sexes), the histopathological changes in the liver were accompanied by increases in the plasma levels of alanine and aspartate aminotransferase (ALAT, ASAT), alkaline phosphatase (ALP), bilirubin and bile acids, and decreases in total protein and albumin. To a lesser extent, a decrease in total protein and increases in plasma enzymes were also observed 150 mg/kg (enzymes were increased in a few animals). Additionally, fasting glucose was decreased in females at 150 and 500 mg/kg. The increased ASAT and ALP values in a few females at 50 mg/kg were not corroborated by histopathological changes and therefore considered to be non-adverse.

Microscopic examination of the spleen showed a treatment-related decrease in the incidence of extramedullary hematopoiesis in both sexes and a slightly increased severity of pigmentation in females at 500 mg/kg. In the absence of adverse changes in haematology parameters, these findings were considered to be non-adverse.

Males treated at 500 mg/kg had lower weights of the prostate gland, Cowper’s gland and ani plus bulbocavernosus muscle complex (LABC). These treatment-related organ weight changes occurred in the absence of macroscopic or microscopic correlate and were therefore considered not to be adverse. Females at 500 mg/kg had lower thymus weights, which correlated with a reduced size of the thymus in one animal. In the absence of a microscopic correlate, the decrease in thymus weight was regarded as non-adverse.

The serum level of thyroid hormone T4 was decreased by about 20% in males at 500 mg/kg. The weight or morphology of the thyroid were not affected by treatment. Therefore, the decrease in T4 was regarded as non-adverse.

Prothrombin time (PT) was increased in males at 500 mg/kg. There was no evidence of impaired coagulation. Possibly, the prolonged PT was associated with the adverse effect on the liver (decreased hepatic synthesis of clotting factors).

 

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg).

 

Developmental results:

At PND 13, body weights of male and female pups were decreased at 150 and 500 mg/kg (by about 10 and 15%, respectively). Pup body weights at PND 1 were similar across the groups, indicating that the lower weights at PND 13 resulted from reduced post-natal growth.

The serum level of thyroid hormone T4 was decreased in male and female pups at 500 mg/kg (on average by about 25-30%).

These developmental effects were considered to be toxicologically relevant. They occurred in the presence of maternal toxicity. No developmental toxicity was observed in the absence of maternal toxicity.

Effects on developmental toxicity

Description of key information

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with formaldehyde, oligomeric reaction products with acetone and diphenylamine in rats by oral gavage.

Parental NOAEL: 50 mg/kg (based on adverse microscopic alterations in the liver and associated changes in clinical pathology parameters in both sexes, and reduced body weight gain and food consumption during lactation in females at the higher dose levels).

Reproduction NOAEL: at least 500 mg/kg.

Developmental NOAEL: 50 mg/kg (based on reduced post-natal growth starting at 150 mg/kg, accompanied by a decrease in serum T4 at 500 mg/kg).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental results:

At PND 13, body weights of male and female pups were decreased at 150 and 500 mg/kg (by about 10 and 15%, respectively). Pup body weights at PND 1 were similar across the groups, indicating that the lower weights at PND 13 resulted from reduced post-natal growth.

The serum level of thyroid hormone T4 was decreased in male and female pups at 500 mg/kg (on average by about 25-30%).

These developmental effects were considered to be toxicologically relevant. They occurred in the presence of maternal toxicity. No developmental toxicity was observed in the absence of maternal toxicity.

Justification for classification or non-classification

The substance is not classified for reproductive/developmental toxicity in accordance with the CLP Regulation. The developmental toxicity observed in the OECD 422 study occurred in the presence of maternal toxicity. No developmental toxicity was observed in the absence of maternal toxicity.