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Diss Factsheets

Administrative data

Description of key information

A No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg body weight/day for repeated dose toxicity was established from an oral sub-acute toxicity study according to OECD guideline 407 with ZINN(II)-RICINOLEAT. 

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-07-26 to 2000-08-23 (inlife phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
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
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System : Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EC).
Source : Charles River Deutschland, Sulzfeld, Germany.
Age at Start of Treatment : Approximately 6 weeks.
Number of animals : 20 males, 20 females (females were nulliparous and non-pregnant).
Randomisation : Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ±
20% of the sex mean.
Identification : Earmark and tattoo.
Conditions : A controlled environment was maintained in the room with optimal conditions of approximately 15 air changes per hour, a temperature of 21 ±3°C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day. Deviations from these optimal conditions occurred, but were considered not to have affected study integrity.
Accommodation : Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight inMacrolon plastic cages with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding. Results of bedding analyses for contaminants are examined and archived.
Diet : Free access to standard pelleted laboratory animal diet (from Carfil Quality BVBA, OudTurnhout, Belgium). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
Water : Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Aliquots of the test substance were taken, weighed and kept until use at the same storage conditions as the original container. The test substance was dosed undiluted as delivered by the sponsor.
Since the test substance was administered as such, chemical analysis of dose preparations was not performed.

Control animals received water (Milli-U) at a dose level of 1000 mg/kg bw/day.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
Method : Oral gavage, using a stainless steel stomach tube ..
Frequency : Once daily for at least 28 days, approximately the same time each day, 7 days per week. Animals were dosed up to the day prior to
necropsy.
Dose volume : Dose volume (ml/kg body weight) was calculated as follows: Dose level (g/kg) / specific gravity of test substance (g/ml). Actual dose volumes were calculated weekly according to the latest body weight.


Frequency of treatment:
Once daily for at least 28 days
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
330 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale : The dose levels were selected on the basis of a 5-day dose range finding study (NOTOX Project 294165).
- Rationale for animal assignment (if not random): Randomisation - Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
- Rationale for selecting satellite groups: none
- Post-exposure recovery period in satellite groups: none
- Section schedule rationale (if not random):
Positive control:
No positive control
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.
- Once daily. detailed dinical observations were made in all animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to start of treatment and on days 8, 15. 22 and 28

BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8,15,22 and 28

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly.


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Subjecttve appraisal wasmaintained during the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (identity)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: refer to table below.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Immediately prior to scheduled post mortem examination
- Animals fasted: Yes, overnight
- How many animals: all
Parameters checked: refer to table below.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: hearing ability, pupillary reflex. static righting reflex and grip strength (Score 0: normal/present,
Score 1 =abnormal/absent).
motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system,
Pearson Technical Services, Debenharn, Stowmarket. England).

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
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 when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
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. No statistical analysis was performed on motor activity data. 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.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of high dose females were slight reduced in week 2, however, there was no statistical significant difference in weight gain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Sligthtly reduced food consumtion in high dose females in week 2
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):
Increased white blood cell counts among high dose males
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Decreased calcium values among high dose males, and increased alanine aminotransferase activity among high dose males and females.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
No mortality occurred during the study period. There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment. Incidental findings that were noted included scabs, alopecia and brown staining of various body parts. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. Salivation, as noted among high dose animals, was considered to be related to multiple intra-oesophageal intubation and/or irritant or bad taste of the test substance. Therefore, these signs were considered of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN:
Body weights of high dose females were reduced in week 2 (189 vs. 202g in control animals). Body weights and body weight gain of other treated animals did not show toxicologically significant changes over the 4-week study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption and relative food consumption (i.e. after correction for body weights) was reduced among high dose females in week 1. There were no other toxicologically relevant differences in food consumption before or after allowance for body weight between treated and control animals.

HAEMATOLOGY:
White blood cell counts were increased among high dose males (pOther haematological parameters were considered not to have been affected by treatment. Minor statistically significant differences arising between controls and treated animals were considered to have occurred as a result of slightly high or low control values or to have arisen by chance. Moreover, a dose-response relationship was absent. Therefore, these changes were considered not to represent a change of toxicological significance.

CLINICAL CHEMISTRY:
Alanine aminotransferase activity was increased among both high dose males (p<0.05) and females (pCreatinine values were decreased among males and females dosed at 330 and 1000 mg/kg/day. In males, this decrease did not show a clear dose-related response. Average creatinine values of males remained within the range expected for untreated rats of this age and strain. Moreover, there was no indication of decreased muscle mass among the animals. Aspartate aminotransferase activity values of two high dose females (animals 36 and 37) were slightly high when compared to historical data. Apart from myositis of the oesophagus (also seen among the other high dose females), microscopic examination did not reveal a possible cause.
There were no other differences noted between control and treated rats that were considered to be related to treatment with test substance. Values in treated animals achieving a level of statistical significance when compared to controls, were considered to be of no toxicological significance since a treatment-related distribution was absent.

NEUROBEHAVIOUR:
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with test substance, when compared to control animals. The variation in motor activity did not indicate a relation with treatment.

ORGAN WEIGHTS:
Changes in organ and organ:body weight ratios were not indicative of a toxic effect of the test substance.
Kidney:body weight ratios were increased among males dosed at 330 and 1000 mg/kg. Absolute kidney weights of males did not show a dose related increase. Since supportive micro and macroscopic effects in the kidneys were absent, this finding was considered to be of no toxicological relevance. Reduced heart weights of high dose females may be related to slightly low terminal body weights. Other organ weights and organ:body weight ratios were considered
to be similar to those of control animals.

GROSS PATHOLOGY:
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings among control or treated animals included dark red discolouration of the thymus, reduced size of the thymus, pale discolouration of salivary glands and an accentuated lobular pattern of the liver. These findings are occasionally seen among rats used in these types of study and were considered changes of no toxicological significance. Watery fluid in the uterus, found in one control female, is related to a stage in the oestrous cycle and is a normal finding.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
An increased incidence of minimal/slight myositis in the oesophagus of high dose animals compared to control animals was considered to be related to the compound administration procedure, rather than a direct effect of the test compound. Other microscopic findings recorded were considered to be within the normal range of background alterations which may be seen in untreated animals of this age and strain.
Dose descriptor:
NOEL
Effect level:
330 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Body weights (gram) summary males and females

 

Treatment

 

Group 1 Control

Group2      

100 mg/kg

Group3    

330 mg/kg

Group4    

1000 mg/kg

Males

Day 1

Mean

209

208

209

211

Week 1

ST. DEV.

6.7

4.2

7.6

5.9

 

N

5

5

5

5

Day 8

Mean

268

266

263

264

Week 2

ST. DEV.

16.1

11.7

23.1

9.0

 

N

5

5

5

5

Day 15

Mean

315

306

312

316

Week 3

ST. DEV.

24.7

20.7

39.1

18.8

 

N

5

5

5

5

Day 22

Mean

352

339

343

341

Week 4

ST. DEV.

26.2

28.7

52.4

28.4

 

N

5

5

5

5

Day 28

Mean

366

357

361

351

Week 4

ST. DEV.

28.6

30.2

65.6

31.6

 

N

5

5

5

5

Females

Day 1

Mean

175

172

175

170

Week 1

ST. DEV.

4.8

4.0

6.4

4.5

 

N

5

5

5

5

Day 8

Mean

202

193

197

189 *

Week 2

ST. DEV.

7.7

4.7

11.1

4.0

 

N

5

5

5

5

Day 15

Mean

213

213

217

209

Week 3

ST. DEV.

10.2

6.1

14.4

9.8

 

N

5

5

5

5

Day 22

Mean

229

223

234

221

Week 4

ST. DEV.

10.9

6.8

21.5

12.5

 

N

5

5

5

5

Day 28

Mean

240

236

238

223

Week 4

ST. DEV.

7.9

11.7

18.5

10.3

 

N

5

5

5

5

 

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

Conclusions:
The findings noted at 1000 mg/kg were without corroborative findings and therefore not considered adverse, the No Observed Adverse Effect Level (NOAEL) was established to be 1000 mg/kg body weight/day.
Executive summary:

In a subacute 28-day oral toxicity study according OECD guideline 407 and EU method B.7, the test substance ZINN(II)-RICINOLEAT (unchanged, no vehicle) was administered to 5 male and 5 female Wistar rats/dose/ group by daily oral gavage at dose levels of 0, 100, 330, and 1000 mg/kg bw/day.

 

RESULTS - Treatment related findings observed were as follows:

100 mg/kg/day: No treatment-related findings noted.

330 mg/kg/day: No treatment-related findings noted.

1000 mg/kg/day: - Body weights reduced (females).

- Food consumption reduced in week 1 (females).

- White blood cell (WBC) counts increased (males).

- Alanine aminotransferase activity ALAT) increased (males/females)

- Calcium values decreased (males)

 

The increased ALAT values in males and females are without any corroborative findings in other liver enzyme parameters, liver histopathology or organ weights.

Elevated WBC counts in male rats are without any corresponding findings, normal distribution of differential leucocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes and Monocytes; percentages of WBC) was observed.

Findings on female body weight and food consumption were mild and observed only in week one of study, companioned with normal body weight gain, they are considered of no toxicological relevance.

From the results presented a No Observed Effect Level (NOEL) for ZINN(II)-RICINOLEAT of 330 mg/kg/day was established.

Based on the absence of functional or morphological disturbances supporting the changes noted for blood parameters, food consumption and body weights in high dose animals, a NOAEL of 1000 mg/kg/day was established.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study was conducted according to modern regulatory standards and was adequately reported.

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 a subacute 28-day oral toxicity study according OECD guideline 407 and EU method B.7, the test substance ZINN(II)-RICINOLEAT (unchanged, no vehicle) was administered to 5 male and 5 female Wistar rats/dose/ group by daily oral gavage at dose levels of 0, 100, 330, and 1000 mg/kg bw/day.

 

RESULTS - Treatment related findings observed were as follows:

100 mg/kg/day: No treatment-related findings noted.

330 mg/kg/day: No treatment-related findings noted.

1000 mg/kg/day: - Body weights reduced (females).

- Food consumption reduced in week 1 (females).

- White blood cell (WBC) counts increased (males).

- Alanine aminotransferase activity ALAT) increased (males/females)

- Calcium values decreased (males)

 

The increased ALAT values in males and females are without any corroborative findings in other liver enzyme parameters, liver histopathology or organ weights.

Elevated WBC counts in male rats are without any corresponding findings, normal distribution of differential leucocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes and Monocytes; percentages of WBC) was observed.

Findings on female body weight and food consumption were mild and observed only in week one of study, companioned with normal body weight gain, they are considered of no toxicological relevance.

From the results a No Observed Effect Level (NOEL) for ZINN(II)-RICINOLEAT of 330 mg/kg/day was established.

Based on the absence of functional or morphological disturbances supporting the changes noted for blood parameters, food consumption and body weights in high dose animals, a NOAEL of 1000 mg/kg/day was established.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Data from a GLP compliant guideline study with reliability 1.

Justification for classification or non-classification

According to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008 there is no need for classification of ZINN(II)-RICINOLEAT for repeated dose toxicity, considering the evaluated effects from a sub-acute toxicity study.