Registration Dossier

Administrative data

Description of key information

A 28-day oral gavage toxicity study resulted in a NOAEL of 50 mg/kg bw based on severe irritating effects of the test substance in the stomach. In a two generation feeding study on rats no such effects became obvious. The NOAEL in this study was 57 mg/kg in males and 71 mg/kg bw for females based on reduced body weight gain. In a 5-day rat pilot study with dermal application of the test substance severe skin reactions were seen starting after the third application or later. These effects may likely to be related to the skin sensitizing capacity of the test substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
other: subacute
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October-November 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MHW-MITI (1986)
Deviations:
not specified
Principles of method if other than guideline:
A 28 day oral gavage study was performed with the test item in maize oil on 5 male and 5 female CD rats per dose at dosages of 0, 10, 50 and 200 mg/kg bw/day according to OECD 407 in 1993. Satellite groups of 5 rats/sex/dose were simultaneously treated with 0 (vehicle alone) and 200 mg/kg bw/day to assess the reversibility of effects.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
maize oil
Details on oral exposure:
Method of administration: gavage (5ml/kg bw)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: daily
Remarks:
Doses / Concentrations:
10, 50, 200 mg/kg bw
Basis:
other: nominal in maize oil
No. of animals per sex per dose:
5 per sex per dose plus
5 animals/sex of the control and high dose group were treated for 28 days and then kept for a 14-day recovery period.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
3 rats/sex and dose were treated with 200, 500, or 1000 mg/kg bw and day for 7 consecutive days.
A single male treated at 1000 mg/kg bw/d and a single female rat treated at 500 mg/kg bw/d were underactive, ungroomed, piloerect and cold on day 1. There were both found dead on the following day.
The remaining rats treated at 1000 mg/kg were sacrificed at day 3 for humane reasons, since they showed the same signs as the animals described above including body weight loss.
In the 500 mg/kg bw group piloerection, post dose salivation etc. were seen from day 1 until termination. The majority of the animals lost bw during the first 3 days of treatment, but gained weight satisfactorily thereafter.
At 200 mg/kg and day piloerection, post-dose salivation and ungroomed conditions were seen. The animals achieved satisfactory body weight gains.
Accordingly the doses for the main study were chosen with 10, 50 and 200 mg/kg bw, the latter being considered to be at or close to the maximum tolerated soage under these conditions of treatment.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of treatment and twice weekly afterwards

FOOD CONSUMPTION : Yes
Food consumption ratio: yes

WATER CONSUMPTION: Yes (visually)

OPHTHALMOSCOPIC EXAMINATION: No (not required at the time of study)

HAEMATOLOGY: Yes

Blood CHEMISTRY: Yes

URINALYSIS: Yes

NEUROBEHAVIOURAL EXAMINATION: No (not required at the time of study)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (kidneys, liver spleen, adrenals, heart, for all animals of the main groups and the reversibility groups; addtionally the stomach was histopathologically investigated since abnormal findings were obsered in this organ)
Statistics:
Inter-group differences in group mean bodyweight change, haematology, blood chemistry and Quantitative urinalysis were evaluated by Student's 't'-test using a pooled error variance. The results of this test are not presented for eosinophil, basophil or monocyte counts where the data are clearly not normally distributed. For organ weights, homogeneity of variance was tested using Bartlett's test. If this was found to be statistically significant, a Behrens-Fisher test was used to perform pairwise comparisons, otherwise Dunnett's test was used. Inter-group differences in the incidence of macro- or micropathological lesions were assessed by the Fisher Exact Probability test. This is in line with current practice but differs from the protocol. Two-tailed analyses were undertaken unless otherwise indicated.
Levels of statistical significance were chosen as p < 0.05 (a), p < 0.01 (b) and p < 0.001 (c) for Student's 't'-test and p < 0.05 (a) and p < 0.01 (b) for Dunnett's or Behrens-Fisher tests and the Fisher Exact Probability test. Inter-group differences that were not statistically significant (p > 0.05) are not annotated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Decedents: Signs included hunched posture, ungroomed appearance, piloerection, and single observations of loose faeces, pallor of eyes, salivation, hypothermia and prostration.
Survials: Signs of reaction to treatment in surviving animals treated at 200 mg/kg/day were minor and generally transient: salivation from Day 5, ungroomed fur on occasions from Day 6, isolated incidences of loose faeces on Days 10 and 11 and piloerection on Days 24 and 25. One male rat had rales between Days 12 and 14 and also showed piloerection and hunched posture on Day 12.
Signs of reaction to treatment in animals treated at 50 mg/kg/day were similarly minor: salivation fro Day 6 and isolated incidences of piloerection, ungroomed fur, hunched posture, rales and laboured respiration.
Three ats treated at 10 mg/kg/day showed salivation on Days 25 and 30.
Mortality:
mortality observed, treatment-related
Description (incidence):
Four males and one female treated at 200 mg/kg/day were found dead or humanely killed between Days 9 and 26.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The bodyweight gains at 200 mg/kg/day were slightly lower than those of the control animals during the first two weeks of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption at 200 mg/kg/day were slightly lower than those of the control animals during the first two weeks of treatment.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day a commensurate reduction in food utilization efficiency was recorded.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Aspartate amino-transferase activities and plasma urea concentrations of male rats treated at 200 mg/kg/day and killed after four week of treatment ere slightly higher than those of control males. These differences were not observed at the end of the two week recovery period.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinary protein concentrations of two male rats treated at 200 mg/kg/day an killed after four weeks of treatment were noticeably higher than those of control rats, Reducing substances and glucose were detected in the urine of further two male rats treated at this dosage. These differences were not observed ten days after treatment had ceased.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights were considered to have been unaffected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no macropathological finding amongst animals killed after four weeks of treatment. Thickened stomach wall was recorded for one female rat which received 200 mg/kg/day and was killed after two weeks cessation of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyperkeratosis an acanthosis in the keratinised region of the stomach were observed in animals treatet at 200 mg/kg/day. All decedents and most animals receiving this dosage and killed after four weeks of treatment showed these findings. Although the findings were still evident in animals two weeks after treatment had ceased, their frequency was much reduced.
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality: Four male and one female animal from the high dose group were foud dead or humanely killed. Ante mortem signs were
rales, gasping and abdominal distension, hunched posture and piloerection. Histopathology revealed that all of these animals had hyperkeratosis and acanthosis of the keratinised region of the stomach and one male animal had accomanying ulceration. The ill-health or demise of these animals was therefore considered to be, at least in part, caused by an irritant effect of the test material on the stomach wall.
Clinical signs:The surviving animals of the high dose group showed salivation, ungroomed appearance and piloerection. The mid-dose animals showed salivation and isolated incidences of pilo-erection, hunched posture and ungroomed fur. Of the low dose group three rats showed salivation.
Body weight: slightly reduced at 200 mg/kg bw
Laboratory findings: Haematology was unaffected. In the high dose group aspartate asmino-tranferase activity and plasma urea concentration were slightly higher in the animals killed at day 29. These differences were not observed at the end of the 2-week reversibility period.
Effects in organs: No treatment related effects on organ weights or macropathological effects observed. Thickened stomach was recorded for one female rat in the high dose group which was humanely killed.
Histopathology: In the rats from the high dose level, hyperkeratosis and acanthosis in the keratinised region of the stomach were seen. Although the findings were still evident in animals 2 weeks after treatment had ceased, their frequency was much reduced.
Hyperkeratosis and acanthosis of the stomach were associated with the repeated gavage adiministration of the test compound. The presence of these findings in the high dosage group is likely to be the result of an irritant effect of the test material rather than a toxic effect. Inflammatory effects occured more prominent in the stomach of high dosed animals.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on irritating effects in the stomach and its consequences at 200 mg/kg bw/day
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The NOAEL in this study is 50 mg/kg bw/day based on irritating effects in the stomach and its consequences at 200 mg/kg bw/day. The NOEL was considered to be very close to this dosage.
Executive summary:

A 28 day oral gavage study was performed with the test item in maize oil on 5 male and 5 female CD rats per dose at dosages of 0, 10, 50 and 200 mg/kg bw/day according to OECD 407 in 1993. Satellite groups of 5 rats/sex/dose were simultaneously treated with 0 (vehicle alone) and 200 mg/kg bw/day to assess the reversibility of effects.

The dose of 200 mg/kg bw/day group resulted in death of four males and one female. Clinical signs and histopathology (all of these animals had hyperkeratosis and acanthosis of the keratinised region of the stomach and one had accompanying ulceration) indicated that these deaths were, at least in part, caused by an irritant effect of the test material on the stomach wall. Most survivors also showed hyperkeratosis and acanthosis and a higher incidence of acute inflammation of the glandular and keratinised stomach regions compared with contol animals. After two weeks recovery, the proportion of animals affected was much lower. A slightly reduced body weight gain was seen in the high dose group and clinical signs as salivation after dosing, ungroomed appearance and piloerection.

At 50 mg/kg bw/day the clinical signs were similar but minor and nearly dissapeared at 10 mg/kg bw.

Hyperkeratosis and acanthosis of the stomach were associated with gavage administration of the test compound. The presence of these findings in the high dosage group is likely to be the result of an irritant effect of the test material rather than a toxic effect. The NOAEL in this study is 50 mg/kg bw/day based on irritating effects in the stomach and its consequences at 200 mg/kg bw/day. The NOEL was considered to be very close to this dosage.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
other: 2-generation reproduction toxicity study
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 2-Generation Study with reliability 1 used as supporting study for repeated dose toxicity
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
Twenty-eight Wistar rats, Crl:WI(WU)/sex/group received diets containing PERKALINK 900 at constant concentrations from the start of the study, during the premating period of at least 10 weeks, mating, gestation and lactation until sacrifice over two successive generations. Dams were allowed to raise one litter and pup data were collected. F0-and F1-dams were sacrificed at or shortly after weaning and a thorough necropsy was performed. F0- and F1-males were sacrificed after at least 11 weeks of exposure. A thorough necropsy was performed. Organs and tissues collected at necropsy were microscopically examined.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar outbred Crl:WI (WU)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 5-6 weeks
- Weight at study initiation: (P) Males: 151.12-155.19 g; Females: 104.13-106.70 g; (F1) Males: 36.87-62.48 g; Females: 37.10-59.40 g
- Housing: Macrolon cages with wood shavings (Lignocel, Type 3/4) as bedding material and strips of paper (Enviro-dri) as environmental enrichment; 4/sex/group during premating period, mated females were housed individually
- Water: ad libitum; domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC)
- Diet: ad libitum; cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
Experimental diets were prepared by mixing powdered Rat & Mouse No. 3 breeding diet, RM3 with the appropriate amounts of test substance. The
diets were mixed in a mechanical blender (Lödige, Paderborn, Germany). During the entire study, fresh batches of experimental diets were prepared approx. once every 2-5 weeks. The experimental diets were stored in a freezer (<-18°C). The feed in the feeders was replaced with fresh portions once a week, and filled up when necessary.

Homogeneity analyses were performed four times during the analytical part of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Principle
The concentration of the substance in RM3 diet was determined by extraction of diet samples with acetonitrile. After shaking and centrifugation, an aliqout of the clear supernatant was diluted woth acetonitrile/methanol/MilliQ water 40/10/50 v/v% and the diluted extract was analysed using HPLC with UV detection at 218 nm. Quantification was obtained by comparison of the peak area of the substance in the sample extracts with those in calibration solutions containing known amounts of the test substance.

Validation criteria
Before analysis of study samples, the analytical method was validated by analysing three spiked samples per dose level, to conform to the following criteria:
- Linearity: the correlation coefficient of the calibration curves should be greater than or equal to 0.996;
- Recovery: the recovery of the test substance from test diet should be between 80% and 110% at each of the concentrations tested;
- Repeatability: the relative standard deviation in the percentage recovery of the three spiked diet samples per concentration level should be less than 10%.

Chromatography
The HPLC-UV conditions were as follow:
HPLC: Waters Alliance
Flow: 1 ml/min
Mobile phase: Acetonitrile/methanol/MilliQ water 40/10/50 v/v%
Gradient: Isocratic
Column: Phenomenex Luna C18, 5 µm, 250 x 4.6 mm
Column temperature: 25 ºC
Injection volume: 50 µl
Detection: DAD (218 nm)
Duration of treatment / exposure:
Males: exposure during the premating (10 weeks) and mating period (maximal 2 weeks): termination after about 12 weeks of treatment
Females: exposure during the premating period of at least 10 weeks, during mating (2 weeks), gestation (4 weeks) and lactation (3 weeks) until sacrifice over two successive generations. Termination for P-females: after about 18 weeks of treatment
Frequency of treatment:
continuously
Remarks:
Doses / Concentrations:
0, 800, 1600 and 3200 ppm in the diet
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
about 57, 120 and 237 mg/kg bw/day
Basis:
other: actual ingested (males)
Remarks:
Doses / Concentrations:
about 71, 139 and 269 mg/kg bw/day
Basis:
other: actual ingested (females)
No. of animals per sex per dose:
28
Control animals:
yes, plain diet
Details on study design:
The study was preceded by a dose-range finding study and a palatability study using the same species, strain and route of administration. For detailed description see chapter 'Toxicity to Reproduction'.
Positive control:
none
Observations and examinations performed and frequency:
Parental animals: Observations and examinations
General clinical observations
Each animal was observed daily in the morning hours by cage-side observations. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

Body weight
Body weights of male and female rats were recorded at the start of administration of the test substance, and weekly thereafter during the premating period. Males were weighed once per week during the mating period until sacrifice. Females were weighed once per week during mating and mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 1, 4, 7, 14 and 21 of lactation. In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

Food consumption
Food consumption was measured per cage by weighing the feeders. The results are expressed in g per animal per day and g per kg body weight per day. Food consumption of male rats was measured twice weekly, except during the mating period. Food consumption of female rats was measured twice weekly during the premating period. Food consumption of mated females was recorded weekly during pregnancy and on day 1, 4, 7, 14 and 21 of lactation.

Intake of the test substance
The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentration of the test substance, the food consumption and the mean body weight measured at the beginning and the end of the pertaining period.

Estrous cyclicity (Parental animals)
Vaginal smears to evaluate the estrus cycle length and normality were made daily for about 3 weeks prior to mating. Smears were made and stained of all females, but only the smears of the control group and the high-dose group were evaluated. No treatment-related changes were observed in the high-dose group; therefore the evaluation of vaginal smears was not extended to the intermediate-dose groups.

Sperm parameters (Parental animals)
Epididymal sperm motility, count and morphology
At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all males of all groups. For this purpose, the cauda epididymis was dissected, weighed, and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for all males of all groups, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained for all males, but only the smears of the control and the high-concentration group males were examined for morphology as no treatment-related changes were observed in the high-dose group.

Testicular sperm count
At necropsy, the left testis of all males of all groups were placed on dry ice and subsequently stored in a freezer (<-70°C) for later determination of the number of homogenization-resistant spermatids. The testes to be analysed were thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma were weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated. The evaluation of homogenization-resistant spermatids was performed in the control group and high-concentration group as no treatment-related changes were observed in the high-dose group.
Sacrifice and pathology:
Postmortem examinations (Parental animals)
Gross necropsy and histology of parental animals
All surviving male and female parent rats were euthanized by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological changes. A necropsy was also performed on animals that died intercurrently (if not precluded by autolysis) or that were killed because they were moribund.

Male animals were sacrificed after mating at approx. 11-12 weeks after the start of the administration of the test substance.
Female animals were sacrificed:
- females with a litter, at or shortly after day 21 of lactation
- females with no viable pups, shortly after the death of the last pup.
- non-pregnant females, 21-26 days after copulation
- non-mated females, approx. 3-4 weeks after the end of the mating period

Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde except for the testes which was preserved in Bouin's fixative:
- adrenals
- brain
- epididymides (left cauda which was used for sperm analysis)
- kidneys
- liver
- ovaries
- pituitary gland
- prostate
- seminal vesicles and coagulating glands
- spleen
- testes (right testis was preserved in Bouin’s fixative, the left one was used for sperm analysis)
- thyroid
- uterus (after counting of the implantation sites)
- vagina
- organs and tissues showing macroscopic abnormalities

Microscopic examination was performed on these organs of all rats of the control and high-dose groups. Treatment-related abnormalities were observed in the ovaries, uterus and vagina. In consultation with the sponsor it was decided to extend the examination of these organs to the intermediate groups.
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined.
Statistics:
The results were analyzed using the methods mentioned below. Other statistical tests may be performed when considered appropriate. P< 0.05 was considered as a level of significance.
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- Fisher's exact probability test were used to evaluate the number of mated and pregnant females and females with live pups.
- Number of implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U test.
- Estrus cyclicity were evaluated by Fisher’s exact test (number of acyclic animals and number of animals with prolonged estrus period), ANOVA followed by Dunnetts multiple comparison tests (number of cycles per animal) and Kruskal-Wallis non parametric ANOVA followed by Mann-Whitney U test (length of the longest cycle).
- Sperm parameters were evaluated by ANOVA followed by Dunnetts multiple comparison tests (epididymal and testicular sperm count and numerical sperm motility parameters) or by Kruskal-Wallis non parametric ANOVA followed by Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology).
- Histopathological changes were evaluated by Fisher's exact probability test.
Clinical signs:
no effects observed
Description (incidence and severity):
The clinical signs observed in the animals during the premating, mating, gestation and lactation period were only seen in one or a few animals and were normal for animals of this strain and age.
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males of the P-generation: Mean body weight of the high-dose group was statistically significantly decreased when compared to the control group from week 1 until sacrifice. Mean body weight change of this group was statistically significantly decreased between weeks 0-1, 2-3, 4-7 and 9-10. Mean body weight of the mid-dose group was statistically significantly decreased in weeks 1, 2, 5, and 9. Body weight change of the animals of this group was statistically significantly decreased between weeks 0-1, 4-5 and 9-10. Mean body weights and body weight changes of animals of the low-dose group were comparable to the control group; only an incidental difference was observed in the body weight change in week 4-5.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males of the F0-generation:
Mean food consumption (expressed as g/animal/day) was statistically significantly decreased in the mid-dose group in week 0-1, weeks 4-7 and 9-10 and in the high-dose group during the entire premating period and after mating until sacrifice. Mean food consumption calculated as g/kg bw/day was statistically significantly decreased in the first week of the study in the high-dose group.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The statistically significant decrease in absolute and relative weights of the ovaries and uterus of the females of the high-dose group of the F0- and the F1-generation were considered related to treatment. The other effects observed on absolute and relative organ weights were inconsistent and or considered to be related to the effects on body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F0-generation:
Gross examination at necropsy revealed no exposure-related findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
F0-generation: In male rats, no treatment-related histopathological changes could be detected.
Females
Histopathological analysis revealed that most female rats of the control group were in the pro-estrus or estrus phase (22/28). One rat was in a persistent anestrus phase.
In the low-dose group, the number of rats in a persistent anestrus phase (10/28) was increased in comparison to the control group (1/28). In the mid-dose group, the number of rats in a persistent anestrus phase further increased (17/28).
Of one animal, of the mid-dose group, the reproductive phase could not be determined. The vaginal epithelium of this rat showed slight atrophy, characterised by the presence of 1 layer of low epithelial cells. In addition the uterine lumen was lined by optically clear tall columnar cells, with oval instead of round nuclei.
In the high-dose group, the reproductive cycle could not be determined in the majority of rats (25/28). These rats showed slight to moderate atrophy of the vaginal epithelium, characterised by the presence of 1 layer of low epithelial cells. In addition, the majority of these rats showed slight atrophy of the uterus (19/28) and a uterine lumen that was lined by optically clear tall columnar cells, with oval instead of round nuclei (26/28).
Semi-quantitative analysis indicated that the average number of corpora lutea in the ovaries, was statistically significantly lower in the high-dose group in comparison to average number in the control group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities were observed. The clinical signs observed in the animals during the premating, mating, gestation and lactation period were only seen in one or a few animals and were normal for animals of this strain and age.

BODY WEIGHT AND WEIGHT GAIN
Males of the P-generation: Mean body weight of the high-dose group was statistically significantly decreased when compared to the control group from week 1 until sacrifice. Mean body weight change of this group was statistically significantly decreased between weeks 0-1, 2-3, 4-7 and 9-10. Mean body weight of the mid-dose group was statistically significantly decreased in weeks 1, 2, 5, and 9. Body weight change of the animals of this group was statistically significantly decreased between weeks 0-1, 4-5 and 9-10. Mean body weights and body weight changes of animals of the low-dose group were comparable to the control group; only an incidental difference was observed in the body weight change in week 4-5.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Males of the F0-generation:
Mean food consumption (expressed as g/animal/day) was statistically significantly decreased in the mid-dose group in week 0-1, weeks 4-7 and 9-10 and in the high-dose group during the entire premating period and after mating until sacrifice. Mean food consumption calculated as g/kg bw/day was statistically significantly decreased in the first week of the study in the high-dose group.

ORGAN WEIGHTS
The statistically significant decrease in absolute and relative weights of the ovaries and uterus of the females of the high-dose group of the F0- and the F1-generation were considered related to treatment. The other effects observed on absolute and relative organ weights were inconsistent and or considered to be related to the effects on body weight.

GROSS PATHOLOGY
F0-generation:
Gross examination at necropsy revealed no exposure-related findings.

HISTOPATHOLOGY:
F0-generation: In male rats, no treatment-related histopathological changes could be detected.
Females
Histopathological analysis revealed that most female rats of the control group were in the pro-estrus or estrus phase (22/28). One rat was in a persistent anestrus phase.
In the low-dose group, the number of rats in a persistent anestrus phase (10/28) was increased in comparison to the control group (1/28). In the mid-dose group, the number of rats in a persistent anestrus phase further increased (17/28).
Of one animal, of the mid-dose group, the reproductive phase could not be determined. The vaginal epithelium of this rat showed slight atrophy, characterised by the presence of 1 layer of low epithelial cells. In addition the uterine lumen was lined by optically clear tall columnar cells, with oval instead of round nuclei.
In the high-dose group, the reproductive cycle could not be determined in the majority of rats (25/28). These rats showed slight to moderate atrophy of the vaginal epithelium, characterised by the presence of 1 layer of low epithelial cells. In addition, the majority of these rats showed slight atrophy of the uterus (19/28) and a uterine lumen that was lined by optically clear tall columnar cells, with oval instead of round nuclei (26/28).
Semi-quantitative analysis indicated that the average number of corpora lutea in the ovaries, was statistically significantly lower in the high-dose group in comparison to average number in the control group.
Dose descriptor:
NOAEL
Effect level:
57 other: mg/kg bw/day (i.e. 800 ppm in the diet)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: decreased body weight and reduced food consumption at the next higher dose
Dose descriptor:
NOAEL
Effect level:
71 other: mg/kg bw/day (i.e. 800 ppm in the diet)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: decreased body weight and reduced food consumption at the next higher dose
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, i.e. decreased body weight and reduced food consumption in the mid-and high-dose groups, the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 57 mg/kg bwt/day for parental males and 71 mg/kg bw/day for parental females.
Executive summary:

In a two-generation study performed according to OECD TG 416 male and female rats (28 per sex per dose) were treated with the test material via food for about 12 weeks (males) and 19 weeks (females), respectively. The administered doses of 0, 800, 1600 and 3200 ppm accounted for 0, 57, 120, and 237 mg/kg bw/day in male rats and 0, 71, 139, and 269 mg/kg bw/day in female rats of the parental generation. Mean body weights of the males in the mid-dose (partially) and high-dose group were dose-related and statistically significant decreased. At necropsy no treatment related gross changes were observed in the parental animals. Histopathological analysis of the ovaries, uterus and vagina showed a clear treatment related effect in the high-dose group females Most rats showed atrophy of the vaginal epithelium and atrophy of the uterus. In addition, the uterine lumen was lined by optically clear, tall columnar cells, with oval instead of round nuclei. No histopathological changes were observed in males.

Based on the results of this study, i.e. decreased body weight and reduced food consumption in the mid-and high-dose groups, the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 57 mg/kg bwt/day for parental males and 71 mg/kg bw/day for parental females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
57 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Scientifically acceptable and well documented.

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

A 28 day oral gavage study was performed with the test item in maize oil on 5 male and 5 female CD rats per dose at dosages of 0, 10, 50 and 200 mg/kg bw/day according to OECD 407 in 1993. Satellite groups of 5 rats/sex/dose were simultaneously treated with 0 (vehicle alone) and 200 mg/kg bw/day to assess the reversibility of effects.

The dose of 200 mg/kg bw/day group resulted in death of four males and one female. Clinical signs and histopathology (all of these animals had hyperkeratosis and acanthosis of the keratinised region of the stomach and one had accompanying ulceration) indicated that these deaths were, at least in part, caused by an irritant effect of the test material on the stomach wall. Most survivors also showed hyperkeratosis and acanthosis and a higher incidence of acute inflammation of the glandular and keratinised stomach regions compared with control animals. After two weeks recovery, the proportion of animals affected was much lower. A slightly reduced body weight gain was seen in the high dose group and clinical signs as salivation after dosing, ungroomed appearance and piloerection.

At 50 mg/kg bw/day the clinical signs were similar but minor and nearly disappeared at 10 mg/kg bw.

Hyperkeratosis and acanthosis of the stomach were associated with gavage and bolus administration of the test compound. The presence of these findings in the high dosage group is likely to be the result of an irritant effect of the test material rather than a toxic effect. The NOAEL in this study is 50 mg/kg bw/day based on irritating effects in the stomach and its consequences at 200 mg/kg bw/day. The NOEL was considered to be very close to this dosage.

In a two-generation study performed according to OECD TG 416 male and female rats (28 per sex per dose) were treated with the test material via food for about 12 weeks (males) and 19 weeks (females), respectively. The administered doses of 0, 800, 1600 and 3200 ppm accounted for 0, 57, 120, and 237 mg/kg bw/day in male rats and 0, 71, 139, and 269 mg/kg bw/day in female rats of the parental generation. Mean body weights of the males in the mid-dose (partially) and high-dose group were dose-related and statistically significant decreased. At necropsy no treatment related gross changes were observed in the parental animals. Histopathological analysis of the ovaries, uterus and vagina showed a clear treatment related effect in the high-dose group females. Most rats showed atrophy of the vaginal epithelium and atrophy of the uterus. In addition, the uterine lumen was lined by optically clear, tall columnar cells, with oval instead of round nuclei. No histopathological changes were observed in males.

Based on the results of this study, i.e. decreased body weight and reduced food consumption in the mid-and high-dose groups, the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 57 mg/kg bw/day for parental males and 71 mg/kg bw/day for parental females.

No histopathological effects were seen in the stomach of the animals after uptake of the test substance with the food over several weeks. This further supports the consideration that the effects seen in the 28 day gavage study are a consequence of irritation after bolus gavage administration and are not due to systemic toxicity.

In a 5 day dermal range-finding study with occlusive treatment for 6h/day corrosive effects were observed at the 1000 and 300 mg/kg dose level. Therefore, a new pilot study was conducted using dose levels of 30 and 100 mg/kg body weight/day at the maximized dose volume of 7.5 ml/kg body weight. Again severe local effects were noted. As in the first pilot study no skin effects were seen on the first 2 -3 days of treatment but appeared on day 3 and later. Thus, these effects may likely be related to the sensitisizing capacity of PERKALINK 900 (as indicated in a Guinea Pig Maximisation Test). Treatment of further animals at lower dose levels was deemed not to provide useful information with regard to systemic toxicity.

Justification for classification or non-classification

The test substance induced hyperkeratosis and acanthosis, partly with ulceration in the stomach of rats after 4 week repeated gavage application. Such effects have not been observed in a 2-generation feeding study in rats with treatment duration of >= 11 weeks. Since the substance is a known eye/mucosal membrane irritant, classified with Eye Damage 1, the effects in the stomach are likely related to this property after oral bolus dosing. The test substance is classified according to Regulation 1272/2008, Annex VI with STOT RE 2 (May cause damage to stomach through prolonged or repeated oral exposure.). To comply with Annex VI the substance is listed in IUCLID chapter 2.1 (self-classification) with STOT RE 2 although the scientific assessment leads to non-classification for repeated dose toxicity.

The test substance induced severe local effects after 5 day repeated dermal exposure for 6h a day under occlusive conditions. First effects were seen at day 3 of treatment and thereafter. Since the test substance is shown to be no acute skin irritant in rabbits, these effects may likely be related to the sensitising capacity of the substance (as indicated in a Guinea Pig Maximisation Test), that led to the classification with Skin Sens. 1A. Thus, no further classification is warranted.