Registration Dossier

Administrative data

Description of key information

Current Key study: OECD 422: NOAEL 200 mg/kg bw/day

90-day toxicity study (OECD 408) study on the substance in progress and will become the key study once available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 2012 to 16 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, equivalent to a valid guidelines and the study was conducted under GLP conditions.
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)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, The Netherlands
- Weight at study initiation: 291 - 367 g (males); 189 - 239 g (females)
- Housing: individually in Makrolon type-3 cages with wire mesh tops
- Diet: Pelleted standard Harlan Teklad 2914C ad libitum
- Water: tap water ad libitum
- Acclimatisation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30 - 70%
- Air changes: 10 - 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 6th September 2012: To: 15th October 2012
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighed into a glass beaker and the vehicle added. The mixtures were stirred using a magnetic stirrer and stored at room temperature.

VEHICLE
- Lot/batch no. (if required): BCBG8285V
- Dose Volume: 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed by GC-FID.

Concentration, homogeneity and stability were determined in samples taken after experimental start (see Table 1 in 'Any other information on materials and methods incl. tables').

Concentration and homogeneity were further determined (see Table 2 in 'Any other information on materials and methods incl. tables').
Duration of treatment / exposure:
males: 4 weeks; females: 7 weeks
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group (I)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
700 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: previous non-GLP range-finding study (details below):
14-day dose-range finding oral toxicity (gavage) study was performed to select doses for OECD 422 study.
Discussion and conclusion on 14-day study:
Oral administration to Wistar rats at doses of 100, 300 and 1000 mg/kg, for 14 days resulted in:
Mortality of three males treated with 1000 mg/kg/day, which were found spontaneously dead on treatment days five or six, four females of this dose group, which were found spontaneously dead on treatment days give or seven and one female of this group, which was sacrificed in extremis on day seven.
In males and females treated with 100 mg/kg/day or 300 mg/kg/day of the test item, no mortality, no test item-related clinical signs, no changes in mean absolute and relative food consumption and no differences of toxicological relevance in mean body weights as well as mean body weight gain and no test item related macroscopic findings were noted.
In femlaes treated with 300 mg/kg/day, reduced thymus weight, thymus to body weight ratio and thymus to brain weight ratio was noted with different statistical significances. The mean liver weight, the mean liver to bodyweight ratio and the mean liver to brain weight ratio were also increased in females of this dose group. In females treated with 100 mg/kg/day the mean liver weight, the mean liver to body weight rato and mean liver to brain weight ratio were slightly increased.

Based on the results, doses of 100 and 300 mg/kg/day of the test item could be considered appropriate for subsequent 28-day repeat-dose toxicity and/or reproduction/developmental screening tests in rats. the dose of 1000 mg/kg/day could nt be proposed for a longer term study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: weekly (males); pre-pairing period days 1 - 8 and 8 - 13 (females); gestation days 0 - 7, 7 - 14 and 14 - 21
post coitum, and days 1 - 4 post partum.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the pre-pairing period.
- Anaesthetic used for blood collection: Yes (light isoflurane)
- Animals fasted: Yes
- How many animals: five males and five females from each group
- Parameters checked: erythrocyte count, haemoglobin, hematocrit, mean corpuscular volume, red cell distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, total leukocyte count, differential leukocyte count, platelet count, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the pre-pairing period.
- Anaesthetic used for blood collection: Yes (light isoflurane)
- Animals fasted: Yes
- How many animals: five males and five females from each group
- Parameters checked: glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, akaline phosphatase, gamma-glutamyl-transferase, bile acids, sodium, potassium, chloride, calcium, phosphorus, total protein, albumin, globulin and albumin/globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
At one time during the study (males shortly before sacrifice and females on day 3 or 4 post partum), relevant parameters in a modified Irwin screen test were performed with five parental males and five parental females. Additionally, locomotor activity was measured quantitatively for the same animals using an Activity Monitor. Activity of the animals (based on beam count) was recorded for 10 minute intervals over a 60 minute period.
Sacrifice and pathology:
All animals sacrificed or found dead were weighed and subjected to a detailed macroscopic examination to establish the cause of death. All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

GROSS PATHOLOGY: Yes
- At sacrifice, the testes and epididymides of all parental males were weighed separately. In addition, the following organs were selected from five males and five females from each group and weighed: adrenals, brain, heart, kidneys, liver, thymus and spleen.

HISTOPATHOLOGY: Yes
- The following tissues were examined: prostrate, seminal vesicles with coagulating gland, testes and epididymides (males); ovaries (females); gross lesions, brain, spinal cord, heart, thymus, thyroids and parathyroids, small and large intestines, stomach, liver, kidneys, adrenals, spleen, trachea and lungs, uterus, urinary bladder, lymph nodes, peripheral nerve and bone marrow (males and females).

- Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. If test material-related morphologic changes were detected in organs of high-dose animals, the same organs from mid-dose and low-dose animals were examined to establish a no-effect level if possible. Histological examination of ovaries was carried out on any females that did not give birth. Microscopic examination of the reproductive organs of all infertile males was made.
Statistics:
The following statistical methods were used to analyse food consumption, body weights and reproduction data:
- Mean and standard deviations were calculated.
- The Dunnett-test based on pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied instead of the Dunnett test when the could not be assumed to follow a normal distribution.
- Fisher's exact test was applied if the variables could be dichotomised without loss of information.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One control male was found spontaneously dead on day five and one 200 mg/kg bw/day male was found spontaneously dead on day 11; both deaths were considered to be due to an incidental influx of dosing solution into the respiratory tract. Two 700 mg/kg bw/day males were found spontaneously dead on days five and six of the pre-pairing period.

Two 200 mg/kg bw/day females were found spontaneously dead on days 23 and 24 of the gestation period; the death of one of them was considered to be due to incidental influx of dosing solution into the respiratory tract. Three 700 mg/kg bw/day females were found spontaneously dead on days five, six and seven of the pre-pairing period, one female of this dose group was sacrificed in extremis on day two of the pre-pairing period and another female of this group was sacrificed in extremis on day 18 of the gestation period.

Apart from the deaths considered to be due to incidental influx of dosing solution into the respiratory tract, the death of one 200 mg/kg bw/day female was considered to probably be pregnancy toxemia but this was not considered to be directly attributable to the test material. In the remainder of the animals, the worsened general condition which was caused by the effects on the forestomach and kidneys and the continual stressful condition attributable to treatment with the test material were considered to be the cause of death.

No clinical signs were noted in control and 50 mg/kg bw/day males during the pre-pairing period. Slight breathing noises were noted in a few 200 and 700 mg/kg bw/day males on one or two days of the pre-pairing period. During the pairing period, slightly soft faeces were noted in a few control males on one day. Slight breathing noises were noted in a few 50 mg/kg bw/day males on two days and in some 200 and 700 mg/kg bw/day males on some days of the pairing period.

No clinical signs were noted in control, 50 and 200 mg/kg bw/day females during the pre-pairing period. Slight breathing noises were noted in 700 mg/kg bw/day females on some days of the pre-pairing period. Prostration and hunched posture were noted in some 700 mg/kg bw/day females on single days. Additionally, slightly ruffled fur and visible slight weight loss were seen in 700 mg/kg bw/day females on some days, slight convulsion was noted in one female on on day and slightly decreased activity was noted in a few females on one day of the pre-pairing period.

During the pairing period, no clinical signs were noted in control, 50 and 200 mg/kg bw/day females. Slightly decreased activity, hunched posture, slightly ruffled fur and visible slightly to moderate weight loss were noted in one 700 mg/kg bw/day females on two days. Slight to moderate hair loss was noted in one 700 mg/kg bw/day female on the ventral thorax, the abdomen, the genital region as well as the left and right thigh. Slight breathing noises were also noted in one female during some days of this period. On one day of the pairing period, slight salivation was noted in one female of this dose group.

No clinical signs were noted during weekly observations in males of all groups during the pre-pairing and pairing period. In one 700 mg/kg bw/day female, slightly decreased activity and hunched posture were noted during the pre-pairing period.

BODY WEIGHT AND WEIGHT GAIN
In the males, no statistically significant differences of the mean bodyweights and the mean bodyweight gain of males treated with 50 mg/kg of the test material were noted when compared with the control males during the pre-mating and mating period of the study. The bodyweight gain of males treated with 200 mg/kg was decreased (p < 0.01) on day two. The mean bodyweight of males treated with 700 mg/kg was slightly decreased (p < 0.05 or p < 0.01) during days three to give and the mean bodyweight gain was slightly decreased (p<0.01) com[pared with controls from day two until day 14 of the pre-mating period. This was not noted during the mating period.
In the females, no test material related effects on the mean bodyweights of the females were observed when compared with the controls during the pre-mating, mating, gestation and lactation period.
The mean bodyweight gain of females treated with 700 mg/kg of the test material was slightly decreased compared to the controls during days three to sever (p < 0.05 or p < 0.01) and on days 12 to 14 (p < 0.05 pr p < 0.01) of the pre-mating period. In females treated with 200 mg/kg, a slight decrease (p < 0.05 or p < 0.01) when compared to controls from days 11 to 21 of the gestation period was observed.

FOOD CONSUMPTION
The mean food consumption of 700 mg/kg bw/day males was slightly decreased (p<0.05) when compared to controls during week one and increased (p<0.01) during week two. The mean food consumption of males of the other treated groups showed no statistically significant changes. 700 mg/kg bw/day females showed a tendency of a slightly decreased mean absolute and relative food consumption during the pre-pairing period. During the first week of the pre-pairing period, this decrease of the mean absolute food consumption was statistically significant (p<0.01).

HAEMATOLOGY
Slightly decreased (p < 0.05) mean haemoglobin values were noted in males treated with 700 mg/kg when compared with the controls after 14 days of treatment. Additionally, the mean red cell volume distribution width was slightly increased (p < 0.01) in males of this dose group when compared with the controls. These values were within the historical control range and were therefore considered not to be related to the test material.
The mean count of total leucocytes was slightly decreased with (p < 0.05) or without statistical significance in males treated with the test material. This was without any dose response relationship and was therefore not considered to be related to administration of the test material.
The mean absolute and/or relative basophils were slightly decreased with different (p < 0.05 or p < 0.01) or without statistical significance in all groups treated with the test material. This was also without a dose-response relationship and therefore not considered to be test material related. The mean values of absolute monocytes and large unstained cells were slightly decreased (p < 0.05) in males treated with the test material The mean value of absolute lymphocytes was slightly decreased (p < 0.05) in males treated with 700 mg/kg of the test material when compared to controls. All these values were found to be within the historical control range and was therefore not attributed to the administration of the test material.
In females, slightly decreased haemoglobin values (p < 0.01), slightly decreased mean content of erythrocytes (p < 0.05) and a slightly decreased haematocrit value (p < 0.01) were noted in females treated at 700 mg/kg when compared to the controls after 14 days of treatment. Additionally, the mean red cell volume distribution width was slightly increased (p < 0.01) in females of this dose group. The mean values of absolute and relative basophils were slightly decreased (p < 0.05) in females treated with 700 mg/kg, the mean values of absolute and relative eosinophils were slightly decreased (p < 0.05) in females treated with 200 mg/kg, and the mean values of absolute large unstained cells were slightly decreased (p < 0.05 or p < 0.01) in females treated with the test material when compared to the controls. The mean value of absolute netrophils was increased (p < 0.01) in the 700 mg/kg group when compared with controls. All these values were within the range of the historical control and therefore could not be considered to be test related.

CLINICAL CHEMISTRY
The mean values of bilirubin were decreased (p < 0.01) in males treated with 200 mg/kg or 700 mg/kg of the test material when compared to controls. This did not have a dose-response pattern and was therefore not considered to be test material related.
The mean triglyceride value of males treated with 700 mg/kg was increased (p < 0.01) when compared with controls. This value exceeded the mean standard deviation of the historical control and was therefore considered an effect of the test material.
The mean content of albumin was increased (p < 0.01), the mean content of globulin was decreased (p < 0.01) and due to this the mean albumin to globulin ratio was increased (p < 0.05) in males treated with 700 mg/kg. It could not be excluded that this was a test material effect.
The mean content of potassium was slightly decreased (p < 0.05) in males treated with 700 mg/kg. Slightly decreased (p < 0.01 or p < 0.05) values of the mean cholesterol in males treated with 200 mg/kg or 700 mg/kg were noted when compared with controls. The mean content of calcium was slightly decreased (p < 0.01) in males treated with 700 mg/kg. The values of these three parameters (potassium, cholesterol and calcium) were within the historical control range and therefore were not attributed to effects of the test material.
In females, the mean values of bilirubin were decreased (p< 0.01) at 200 mg/kg or 700 mg/kg. This decrease showed a dose-response relationship and was therefore attributed to the test material
The mean triglyceride value for females treated with 700 mg/kg was increased (p < 0.01) when compared with controls. This value exceeded the mean standard deviation of the historical control range and was also considered to be an effect of the test material.
The mean value of protein was decreased in females at 200 mg/kg or 700 mg/kg (p < 0.05 and p < 0.01) when compared with controls. The mean content of globulin was decreased (p < 0.05 or p < 0.01) in all groups treated with the test material. This increase was marked in females at 700 mg/kg which resulted in an increase in the mean albumin to globulin ratio (p < 0.05) when compared with controls. This could not be excluded as a test material effect.
The mean cholesterol value was slightly decreased (p < 0.05) in 700 mg/kg females. The mean content of calcium was slightly decreased (p < 0.01) and the mean phosphorous content was increased (p < 0.05) in this group also. These values were within the historical control range and were therefore not attributed to effects of the test material.

NEUROBEHAVIOUR
None of the parameters investigated during the functional observational battery gave an indication of a test material-related effect. Mean values of fore hand grip strength of 200 and 700 mg/kg bw/day males were slightly decreased when compared with controls. No differences in mean hind limb grip strength of treated males were noted when compared to controls. No differences in mean fore- and hind limb grip strength of treated females were noted when compared to controls.

No statistically significant effects on locomotor activity of treated males and females were noted.

ORGAN WEIGHTS
No statistically significant changes in the mean organ weights, organ to body weight ratios and organ to brain ratios were noted in females when compared with the control animals.
In males, the mean liver weight, the mean liver to body weight and the mean liver to brain weight ratio were increased (p < 0.01) in animals treated with 700 mg/kg/day of the test item when compared with controls. The mean thymus weight, the mean thymus to body weight ratio and the mean thymus to brain weight ratio were decreased (p<0.01) in males of this dose group when compared with controls. Both findings were considered to be test item related.

GROSS PATHOLOGY
There were no gross lesions in the animals that died as a results of treatment with the test material, and the gross changes were considered to be incidental lesions, non-specific changes that are commonly observed in the decedent the lesions are considered to be related to stressful conditions and also spontaneous lesions which are associated with animals of the strain and age used in the study.
Incomplete collapse of the lung was observed in two animals (11 a control male and 31 a male from the 200 mg/kg group). This correlated with observed alveolar oedema (minimal in animal 11 and moderate in animal 31).
In animal number 31, slight mucosal necrosis was observed microscopically in the trachea. These microscopic changes were considered to be due to incidental influx (e.g. regurgitation) of the dosing solution into the respiratory tract, this was considered to be related to the morbidity of this animal.
In animal number 11, incidental influx of the dosing solution into the respiratory tract was suspected to be the cause of the animals morbidity, although histological changes could not be identified on the trachea die to progressive of autolysis.
Discolouration recorded in lungs, thymus and lymph node, which were correlated microscopically with congestion and/or agonal haemorrhage were considered to be non-specific changes commonly observed in the decedent.
The black-foci of the gastric fundus and the reduced splenic size, which were correlated microscopically with glandular stomach erosion and splicing follicular atrophy/reduced red pulp area respectively, were considered to be non-specific, secondary changes associated with a stressful condition.
Liquid and/or brown contents in the stomach and/or intestines were recorded in animals 40, 42 and 78 (2 males from the 700 mg/kg group and one female from the 700 mg/kg group). Such microscopic findings may be observed when there were haemorrhagic lesions in the alimentary tracts. In animal 78 (the female from the 700 mg/kg group), black-contents in the ileum and cecum were recorded. Although there were no histological abnormalities in these organs, glandular stomach erosion with mucosal regeneration was observed microscopically, and therefore, macroscopic findings in the ileum and cecum were considered to be associated with gastric injuries.
In the other two males from this group, microscopic changes that were considered to be attributable to macroscopic findings could not be identified in these animals due to the progression of autolysis.
Splenic constriction and dark red discolouration of the ovary were correlated with focal fibrosis and congestion respectively when examined microscopically. These were considered to be spontaneous changes which may be observed in rats of this strain and age.
Gross lesions in the survivors attributable to treatment with the test item were recorded in the liver and the thymus.
Enlarged liver was recorded in two males of group 1, one male of group 2 and five males of group 4, the incidence was higher in group 4 males.
Reduced thymic size was observed in four males of group 4.
All other gross lesions recorded in the animals surviving to termination were within the normal background alterations for this age and strain of rat.

HISTOPATHOLOGY: NON-NEOPLASTIC
The test material induced histomorphological changes in testes, epididymides, prostate glands, seminal vesicles, coagulating glands, kidneys, stomach, liver, adrenal glands, thymus and trachea.
Degeneration of spermatocytes/spermatids and increased incidence and/or severity of Sertoli cell vacuolation were recorded in testes of group 4 (700 mg/kg). Increase in cellular debris in the tubules, decrease in elongate spermatids and/or loss of germ cells were frequently observed in the affected testes of males in group 4. No abnormalities were identified on the Spermatogonia. Continual stressful condition of the animals suggested by thymic atrophy and/or adrenocortical hypertrophy was expected mainly in animals of group 4 and might have contributed to the testicular lesions, although the possible effects on the spermocytes/spermatids could not be excluded.
Increased cellular debris in the duct lumen as well as oligospermia in epididymides was recorded in males of group 4 (700 mg/kg). These were considered to be secondary effects of the testicular lesions observed in this group. Reduced secretion was recorded in the prostate glands, seminal vesicle and coagulating glands in males of group 4 as well. There were no indicators of cellular/tissue injuries in these organs, and therefore, these findings were considered to be secondary changes following the testicular lesions, or the poor condition of these animals.
In the females, no specific effects were observed in the ovaries.
In the kidney, increased incidence and/or severity of tubular degeneration/regeneration were recorded in both sexes of group 4 (700 mg/kg), these degenerative changes were considered to be adverse.
In the stomach, hyperkeratosis and/or squamous cell hypertrophy (increased mucosal thickness) of the forestomach were recorded in animals of groups 3 and 4 (200 and 700 mg/kg, respectively). In addition to these findings, squamous cell hyperplasia and ulcer were recorded in the forestomach of the animals that died in group 4. These findings were considered to be attributable to the irritating properties of the test material. Hyperkeratosis and squamous cell hypertrophy of the forestomach were considered to be a simple reactive change to the irritative stimuli of the test material and was therefore not considered to be an adverse effect. The observed ulceration and squamous cell hyperplasia were tissue injury and its reparative process and was therefore considered to be adverse.
In the liver, centrilobular hepatocellular hypertrophy was recorded in both sexes of groups 3 and 4 (200 and 700 mg/kg, respectively). This was considered to be related to metabolism and adaptive in character, and there were no further indications of liver injuries. This was therefore not considered to be adverse.
Epithelial hypertrophy and regenerated mucosal epithelium of trachea were sporadically recorded in the survivors of groups 2 and 4. Mucosal necrosis and regenerated mucosal epithelium were also recorded in the decedent of groups 3 and 4. In addition, a slight increase in the group mean severity of mucosal/submucosal inflammatory cell infiltration was recorded in group 4. These were not caused by the systemic toxicity of the test material and were considered to be related to incidental influx (e.g. regurgitation) of dosing solutions containing the test material into the respiratory tract. The irritating properties of the test material was attributed to the slightly higher incidence in severity of the mucosal injuries in the groups treated with the test material compared to the control group.

HISTORICAL CONTROL DATA (if applicable)
Historical control data was included for comparison of haematolocy and clinical biochemistry parameters.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality; body weight; clinical chemistry; gross pathology; organ weights; histopathology
Critical effects observed:
no

Table 3: Food consumption in the pre-pairing period (g/animal/day)

     0 mg/kg  50 mg/kg  200 mg/kg  700 mg/kg
 Males          
 Days 1 to 8  Mean  21.8  22.8  23.1  16.8*
   S.D.  4.5  1.9  2.2  7.0
   n  11  11  11  11
 Days 8 to 13  Mean  23.9  23.4  23.6  27.3**
   S.D.  1.7  2.3  2.9  2.3
   n  10  11  11  9
 Mean of means    22.9  23.1  23.3  22.1
 Females          
 Days 1 to 8  Mean  17.4  17.2  16.8  12.0**
   S.D.  2.3  1.5  1.4  5.1
   n  11  11  11  11
 Days 8 to 13  Mean  17.5  17.7  17.2  17.8
   S.D.  2.3  1.5  1.2  5.1
   n  11  11  11  8
 Mean of means    17.5  17.4  17.0  14.9

Dunnett's

Table 4: Mean Bodyweights and Bodyweight Gain of Males

Period

Day

0 mg/kg (control)

50 mg/kg

200 mg/kg

700 mg/kg

BW (g)

BW gain (%)

BW (g)

BW gain (%)

BW (g)

BW gain (%)

BW (g)

BW gain (%)

Pre-mating

1

327

0

321

0

323

0

324

0

2

332

1

323

1

321

0**

319

-2**

3

333

2

326

1

326

1

308*

-5**

4

336

3

328

2

328

2

303**

-6**

5

336

4

331

3

332

3

306*

-5**

6

338

4

334

4

333

3

311

-2**

7

338

4

335

4

335

4

314

-1**

8

344

6

338

5

337

4

318

0**

9

344

6

340

6

339

5

321

1**

10

348

7

343

7

342

6

324

2**

11

349

8

344

7

342

6

332

1**

12

352

9

346

8

346

7

324

2**

13

354

9

349

9

350

8

326

3**

14

346

7

342

6

342

5

319

0**

Mating period

1

350

0

344

0

346

0

323

0

2

352

1

347

1

347

0

326

1

3

351

0

349

1

349

1

329

2

4

351

0

349

1

348

1

328

1

5

358

2

355

3

352

2

334

3

6

359

3

356

3

352

2

338

5

7

362

3

358

4

355

3

341

5

8

364

4

360

5

357

3

342

6

9

367

5

362

5

360

4

343

6

10

365

4

363

6

358

4

345

6

11

367

5

367

7

359

4

343

6

12

372

6

371

8

364

5

348

8

13

374

7

372

8

366

6

352

9

14

374

7

373

8

366

6

351

9

15

376

8

376

9

369

7

351

9

BW = Bodyweight

-/**Dunnet-test based on pooled variance * p < 0.05 ** p < 0.01

 

Table 5: Mean Bodyweights and Bodyweight Gain of Females

Period

Day

0 mg/kg (control)

50 mg/kg

200 mg/kg

700 mg/kg

BW (g)

BW gain (%)

BW (g)

BW gain (%)

BW (g)

BW gain (%)

BW (g)

BW gain (%)

Pre-mating

1

213

0

212

0

212

0

212

0

2

214

1

213

0

210

-1

211

-1

3

217

2

214

1

213

1

204

-4**

4

218

2

219

3

216

2

201*

-5**

5

218

3

217

2

216

2

206

-4**

6

219

3

217

3

215

2

206

-3**

7

220

4

218

3

218

3

213

0*

8

222

5

222

5

219

4

216

1

9

221

4

221

4

220

4

217

2

10

222

4

222

5

220

4

213

0

11

225

6

224

6

223

5

217

2

12

226

7

226

7

222

5

214

1**

13

225

6

226

7

224

6

214

1*

14

222

4

223

5

222

5

204

-4**

Gestation period

0

228

0

227

0

233

0

-

-

1

233

2

232

2

236

1

-

-

2

240

5

238

5

241

4

-

-

3

243

6

239

6

244

5

-

-

4

247

8

245

8

247

6*

-

-

5

251

10

249

10

251

8

-

-

6

254

11

253

12

255

10

-

-

7

257

12

255

13

258

11

-

-

8

262

15

260

15

261

12

-

-

9

264

16

263

16

263

13

-

-

10

268

18

266

17

268

15

-

-

11

276

21

273

20

273

17*

-

-

12

280

23

277

22

276

19*

-

-

13

284

25

281

24

280

20*

-

-

14

287

26

285

26

284

22*

-

-

15

294

29

291

28

289

24*

-

-

16

303

33

301

33

296

27*

-

-

17

316

38

312

38

304

31**

-

-

18

329

44

323

43

315

35**

-

-

19

342

50

336

48

326

40**

-

-

20

355

56

348

54

337

45**

-

-

21

364

59

356

57

344

48**

-

-

Lactation period

1

267

0

270

0

269

0

2

268

1

270

0

270

0

3

273

2

273

1

272

1

4

276

4

275

2

272

1

BW = Bodyweight

-/**Dunnet-test based on pooled variance * p < 0.05 ** p < 0.01

 

Conclusions:
Under the conditions of the test, the no-observed-adverse effect level (NOAEL) was considered to be 200 mg/kg/day for males and females, and the no-observed effect level (NOEL) was established as 50 mg/kg/day in both sexes.
Executive summary:

The test material was administered to male rats for at least 28 days and to female rats for 14 days prior to mating, through the mating and gestation periods until the F1 generation reached day 4 post partum. The test material was applied at 0, 50, 200 and 700 mg/kg bw/day (groups 1 to 4, respectively). The following effects were noted:

One male in group one, one male and two females of group three and two males and five females of group four died or were killed in extremis prematurely. The cause of death of the male in group one and one male and one female in group three was considered to be incidental influx of the dosing solution into the respiratory tract. In the other animals, the stressful condition attributable to treatment of the test material was considered to be the cause of death.

None of the parameters investigated during the functional observational battery indicated a test material-related effect.

The mean food consumption of 50 and 200 mg/kg bw/day males were not statistically significant when compared to the controls. The mean food consumption of 700 mg/kg males was slightly decreased in week one and slightly increased in week two when compared to the controls. 700 mg/kg bw/day females showed a tendency of a slightly decreased mean absolute and relative food consumption on the pre-pairing period. During the first week of this period, the decreased of the mean absolute food consumption was statistically significant. In 200 mg/kg bw/day females, the mean food consumption was slightly decreased during the lactation period.

No statistically significant differences in mean body weights and mean body weight gain of 50 mg/kg bw/day males were noted compared to the controls during the pre-pairing and pairing period. The mean body weight of 700 mg/kg bw/day males was slightly decreased during days three to five and the mean body weight gain was slightly decreased when compared to the controls from day two to day 14 of the pre-pairing period (this was not noted during the pairing period). No effects on mean body weights of females were observed during the pre-pairing, pairing, gestation and lactation periods. The mean body weight gain of 700 mg/kg bw/day females was slightly decreased compared to controls during days three to seven and on days 12 to 14 of the pre-pairing period. The mean body weight gain was slightly decreased in 200 mg/kg bw/day females when compared to controls from day 11 to 21 of the gestation period.

No changes in haematology parameters were noted in males and females when compared to the controls.

The mean values of bilirubin were decreased in 200 and 700 mg/kg bw/day males when compared to the controls; this decrease showed a dose-response relationship and is therefore considered to be test material related. The mean triglyceride value of 700 mg/kg bw/day males was increased when compared to the controls; this value exceeds the mean standard deviation of the historical reference data and is therefore considered to be test material related. The mean albumin content was increased, the mean globulin content was decreased and, due to this, the mean albumin to globulin ratio was increased in 700 mg/kg bw/day males; it cannot be excluded that this is test material related. The mean bilirubin values decreased in 200 and 700 mg/kg bw/day females when compared to the controls; this decrease showed a dose response relationship and is therefore considered to be test material related. The mean triglyceride value in 700 mg/kg bw/day females was increased when compared to the controls; this value exceeds the mean standard deviation of the historical reference data and is considered to be test material related. The mean protein value decreased in 200 and 700 mg/kg bw/day females when compared to the controls. The mean content of globulin decreased in all treated groups compared to the controls; due to this the mean albumin to globulin ratio increased in 700 mg/kg bw/day females when compared to the controls and it cannot be excluded that this is test material related.

No statistically significant differences in mean organ weights, mean organ to body weight ratios and mean organ to brain weight ratios were noted in treated females when compared to controls. In 700 mg/kg bw/day males, the mean liver weight, the mean liver to body weight ratio and the mean liver to brain weight ratio were statistically significantly increased when compared to controls. The mean thymus weight, the mean thymus to body weight ratio and the mean thymus to brain weight ratio of this dose group were statistically significantly decreased when compared to controls.

In the kidney, increased incidence and/or severity of tubular degeneration/regeneration were recorded in both sexes at 700 mg/kg bw/day and such degenerative lesions were considered to be adverse. In the stomach, hyperkatosis and/or squamous cell hypertrophy were recorded in 200 and 700 mg/kg bw/day animals. In addition to these findings, squamous cell hyperplasia and ulcer were recorded in the forestomach of 700 mg/kg bw/day animals. These findings were considered to be related to the irritant property of the test material.

In the liver, centrilobular hepatocellular hypertrophy was recorded in both sexes of 200 and 700 mg/kg bw/day animals. This was considered to be of metabolic nature and adaptive character and there were no further indicators of liver injuries. Epithelia hypertrophy and regenerated mucosal epithelium of trachea were sporadically recorded in 50 and 700 mg/kg bw/day survivors. Mucosal necrosis and regenerated mucosal epithelium were also recorded in the decedent of 200 and 700 mg/kg bw/day animals. In addition, a slight increase in the group mean severity of mucosal/submucosal inflammatory cell infiltration was recorded in 700 mg/kg bw/day animals. These were considered to be associated with incidental influx of the dosing solution containing the test material.

Degeneration of spermatocytes/spermatids and increased incidence and/or severity of Sertoli cell vacuolation were recorded in testes of 700 mg/kg bw/day males. Increase in cellular debris in the tubules, decrease in elongated spermatids and/or loss of germ cells were frequently observed in the affected males in this group. No abnormalities were identified in spermatogonia. Continual stressful condition being suggested by thymic atrophy and/or adrenocortical hypertrophy was expected mainly at 700 mg/kg bw/day and might have contributed to the testicular lesions although the possible direct effects of the test material on spermatocytes/spermatids could not be excluded. Degenerative testicular changes recorded in group four animals, irrespective of the cause, were considered to be responsible for female infertility and considered to be adverse. Increased cellular debris in the duct lumen as well as oligospermia in epididymides was recorded in 700 mg/kg bw/day males. These were considered to have been secondary events following testicular lesions. Reduced secretion was recorded in the prostate glands, seminal vesicles and coagulating glands in 700 mg/kg bw/day males. There were no indicators of cellular/tissue injuries in these organs and, therefore, these findings were considered to be secondary changes following the testicular lesions caused by worsened general condition.

The NOAEL was considered to be 200 mg/kg bw/day for both sexes and the NOEL was considered to be 50 mg/kg bw/day for both sexes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

In the OECD 422 study, the test material was dosed at 0, 50, 200 and 700 mg/kg bw/day in a repeated dose and reproductive/developmental toxicity screen. The test material induced test material related mortality in the high dose group and bodyweight (and bodyweight change), food consumption, haematological, clinical chemistry, microscopic and macroscopic changes in male and female rats. Under the conditions of the test, the no-observed-adverse effect level (NOAEL) was considered to be 200 mg/kg/day for males and females, and the no-observed effect level (NOEL) was established as 50 mg/kg/day in both sexes.

Justification for classification or non-classification

According to Regulation EC 1272/2008, the substance does not require classification for repeated dose toxicity.