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Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

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Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From January 20, 1999 to February 17, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
RA study
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
- Based on guidelines
Sex:
male/female
Details on test animals and environmental conditions:
Origin: Harlan Winkelmann
Acclimatization: at least 5 d
Body weight at start for males: 280 g and for females: 209 g
Age: 5-6 weeks
Temperature and relative humidity: 22 +/-3°C and 50+/-20%, respectively
Lighting time: 12 h daily
Food: ssniff R/M-H (V 1534), ad libitum (except for the period in which the animals were kept in diuresis cages)
Water: tap water, ad libitum (except for the period in which the animals were kept in diuresis cages)
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Determination of the solubility, stability and homogenecity of the test substance (3 samples)
- Method: HPLC-separation on a reverse phase column followed by a spectrophotometric detection (515 nm)
- Results obtained: acceptable
Duration of treatment / exposure:
Main test group: 28 d
Recovery group: 14 d
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
On Day 29, five animals from each group were sacrificed and necropsied. The remaining animals from the control and high dose groups were sacrifiecd and necropsied after a recovery period of 14 d.
Volume administered: 5 mL/kg bw/day
Positive control:
-
Observations and examinations performed and frequency:
- Behavior and state of health were observed daily
- Body weights and food consumption were recorded twice weekly, and water consumption once weekly
- Once before the first treatment and thereafter once a week detailed clinical observations were performed in all animals outside the home cage in a standard arena (open field). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa, and impairment of dental growth
- Neurotoxicological measurements including assessment of sensory function, motor activity, forelimb and hindlimb grip strength were conducted at the end of the treatment period
- Hematological examinations and clinical chemistry were carried at the end of the treatment period and after the recovery period
- Urine analysis was performed at the end of the treatment period and after the recovery period
Sacrifice and pathology:
- During necropsy, the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Main organs and tissues were processed for histopathological examination and checked for microscopically visible changes
Clinical signs:
no effects observed
Description (incidence and severity):
- No systemic effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Slightly decreased albumin values in both sexes from the high dose group and slightly decreased protein concentrations in females from the high dose group
- The serum was discolored salmon pink
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
- The urine showed a salmon pink discoloration in all animals from the high group and a very slight salmon pink discoloration in one male from the intermediate dose group
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Kidneys were discolored red in both sexes from the intermediate and high dose group at necropsy. Additionally, light red discoloration of testes and adipose tissue was observed in the animals from the high group
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Mixed cellular infiltrations in the submucosa of the stomach in the animals from the intermediate and high dose group were observed, with the severity being dose-dependent
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOEL
Remarks:
local effects
Effect level:
62.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEL
Remarks:
systemic effects
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Under the study conditions, the NOELs of the substance for local and systemic effects in rats were determined to be 62.5 mg/kg bw/day and 250.0 mg/kg bw/day, respectively.
Executive summary:

A study was conducted to determine the oral repeated dose toxicity of the read-across substance according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. Five Sprague-Dawley rats per sex per group were administered by oral gavage repeated dose of the substance at 0, 62.5, 250 and 1000 mg/kg bw for 28 d followed by a 14 d recovery period. The solubility, stability and homogeneity of the test formulation were analysed by HPLC and spectrophotometric methods and the results were considered acceptable. Clinical signs and mortality were observed daily. Body weights and food consumption were recorded twice weekly, and water consumption was recorded once weekly. Once before the first treatment and thereafter once a week, detailed clinical observations were performed in all animals in an open field. Additionally, the animals were examined for opacity of the eyes, damage to the oral mucosa, and impairment of dental growth. Neurotoxicological measurements including assessment of sensory function, motor activity, forelimb and hindlimb grip strength were conducted at the end of the treatment period. Hematological and clinical chemistry measurements were carried out at the end of the treatment period and after the recovery period. Urine analysis was performed at the end of the treatment period and after the recovery period. During necropsy, the animals were examined for macroscopically visible abnormalities and organ weights. Selected organs and tissues were processed for histopathological examination. No evidence of systemic toxicity was observed except for slightly decreased albumin levels in both sexes and slightly decreased protein concentrations in females from the high dose group. Microscopically, mixed cellular infiltrations in the submucosa of the stomach at the mid and high dose groups were observed, suggestive of localised irritation in the stomach. Under the study conditions, the NOELs of the substance for local and systemic effects in rats were determined to be 62.5 mg/kg bw/day and 250.0 mg/kg bw/day, respectively (Hofmann, 1999).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20 July 2016 - 12 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
A total of 90 Hsd Sprague Dawley SD rats (45 males and 45 virgin females), 6 to 7 weeks old and weighing 175 to 200 g for males and 151 to 175 g for females, were ordered from and supplied by Envigo RMS srl, San Pietro al Natisone (UD), Italy.
After arrival, on 28 July 2016, the weight range for each sex (192-202 g for males, 164-173 g for f emales, slightly outside the range at order for the males) was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of 20 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations. The animals were housed in a limited access rodent facility. Animal room controls were set to mai ntain temperature and relative humidity at 22 °C±2 °C and 40-70% respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
Route of administration:
oral: gavage
Details on oral exposure:
MAIN GROUPS (GROUPS 1 TO 4)
- Males: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and up to the day before necropsy.
- Females: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to Day 3 post partum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in RTC Study No. A2213 in the range from 5 to 125 mg/mL. Linearity, accuracy and precision were within the limits stated in RTC SOPs for solutions (r > 0.98; accuracy 95-105 %; precision CV < 5 %).
Stability was verified in the range from 5 to 125 mg/mL and was found to be at least 24 hours at room temperature and 8 days at 5±3°C. The proposed formulation procedure for the test substance was checked in the range from 5 to 125 mg/mL by chemical analysis (concentration) in RTC Study no. A2213 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (90-110 %). Samples of the formulations prepared on Day 1 and Week 6 were analysed to check the concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for concentration of solutions (90-110 %).
Duration of treatment / exposure:
The test substance was administered daily by oral gavage to male rats for 36 d and to female rats for 41 to 59 d.
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
Each group comprised 10 male and 10 female rats (Groups 1 to 4).
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Parental animals: Observations and examinations
1) Clinical signs: once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of postdose reactions.
2) Observations of the cage tray: observations of the cage tray were performed during mating period for all main groups and were recorded daily.
3) Body weight: males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.
Dams were also weighed on Days 1 and 4 post partum.

Litter observations
The day that offspring were first detected in the cage was considered Day 0 post partum. As soon as possible after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and only live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters.
Sacrifice and pathology:
Postmortem examinations (parental animals)
Parental animals were killed by exsanguination under isofluorane anaesthesia.

1) Parental males: the males were sacrificed after completion of the mating period after 36 days of treatment.
2) Parenteral females: the females with live pups were sacrificed on Day 4 post partum. The females which did not give birth 25 days after positive identification of mating were killed shortly after.
3) Necropsy: The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination
4) Females: all females were examined also for the following: – external and internal abnormalities
4) Organ weights: from all animals completing the scheduled test period, the following organs were dissected free of fat and weighed: adrenal glands, brain (cerebrum, cerebellum, medulla/pons), epididymides, kidneys, liver, ovaries, prostate glands, seminal vescicles with coagulating glands, testes, thyroid, uterus including cervix. The ratios of organ weight to body weight were calculated for each animal.
5) Tissues fixed and preserved: Samples of all the following tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol): abnormalities, adrenal glands, brain (cerebrum, cerebellum, medulla/pons), clitoral gland, epididymides, kidneys, liver, ovaries, penis with preputial gland, prostate gland, seminal vesicles with coagulating glands, spleen, testes, thymus, thyroid, uterus – cervix, vagina.
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.

The examination was restricted as follows:
- tissues specified above from from all animals in the control and high dose group killed at term
- all abnormalities in all groups.

Postmortem examinations (offspring)
All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 4 post partum) were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the ot her parameters. Inter group differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if ‘n’ was more than 5.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Terminal body weight was unaffected by treatment in both sexes. Absolute and relative organ weights did not show any difference.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Animals killed at termination did not show relevant macroscopic changes that could be treatment - related. The changes observed such as red staining of tail, enlarged liver or ovaries and reduced thymus are suggested to be likely due to the colour of the test substance and / or characteristically seen in untreated Sprague Dawley rats of the same age.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment - related changes were noted. The sporadic lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related systemic toxicity
Conclusions:
Under the study conditions, the NOEL (No Observed Effect Level) of the substance for systemic toxicity was considered to be 1000 mg/kg bw/d in rats.
Executive summary:

A study was conducted to determine the repeated dose toxicity of the substance as part of reproductive/developmental screening according to OECD Guideline 421, in compliance with GLP. The test substance was administered daily by oral gavage to male (36 d) and female (from 41 to 59 d) Sprague Dawley SD rats at dose levels of 0, 62.5, 250 and 1000 mg/kg bw/d. The following investigations were performed on parental animals of all groups: mortality check, clinical signs, body weight, food consumption, macroscopic observations and organ weights. Measurement of body weight, clinical signs and macroscopic observations of pups were also performed. Histopathological evaluation of reproductive organs/tissues was performed on all control and high dose males and females, as well as on all abnormalities detected during post mortem observation. As a result of the treatment no signs of systemic toxicity were observed in the parental animals as well as in the pups. Under the study conditions, the NOEL of the substance for systemic toxicity was considered to be 1000 mg/kg bw/d (Longobardi, 2017).

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

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral repeated dose toxicity:

A study was conducted to determine the oral repeated dose toxicity of the read-across substance according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. Five Sprague-Dawley rats per sex per group were administered by oral gavage repeated dose of the substance at 0, 62.5, 250 and 1000 mg/kg bw for 28 d followed by a 14 d recovery period. The solubility, stability and homogeneity of the test formulation were analysed by HPLC and spectrophotometric methods and the results were considered acceptable. Clinical signs and mortality were observed daily. Body weights and food consumption were recorded twice weekly, and water consumption was recorded once weekly. Once before the first treatment and thereafter once a week, detailed clinical observations were performed in all animals in an open field. Additionally, the animals were examined for opacity of the eyes, damage to the oral mucosa, and impairment of dental growth. Neurotoxicological measurements including assessment of sensory function, motor activity, forelimb and hindlimb grip strength were conducted at the end of the treatment period. Haematological and clinical chemistry measurements were carried out at the end of the treatment period and after the recovery period. Urine analysis was performed at the end of the treatment period and after the recovery period. During necropsy, the animals were examined for macroscopically visible abnormalities and organ weights. Selected organs and tissues were processed for histopathological examination. No evidence of systemic toxicity was observed except for slightly decreased albumin levels in both sexes and slightly decreased protein concentrations in females from the high dose group. Microscopically, mixed cellular infiltrations in the submucosa of the stomach at the mid and high dose groups were observed, suggestive of localised irritation in the stomach. Under the study conditions, the NOELs of the substance for local and systemic effects in rats were determined to be 62.5 mg/kg bw/day and 250.0 mg/kg bw/day, respectively (Hofmann, 1999).

A study was conducted to determine the repeated dose toxicity of the substance as part of reproductive/developmental screening according to OECD Guideline 421, in compliance with GLP. The test substance was administered daily by oral gavage to male (36 d) and female (from 41 to 59 d) Sprague Dawley SD rats at dose levels of 0, 62.5, 250 and 1000 mg/kg bw/d. The following investigations were performed on parental animals of all groups: mortality check, clinical signs, body weight, food consumption, macroscopic observations and organ weights. Measurement of body weight, clinical signs and macroscopic observations of pups were also performed. Histopathological evaluation of reproductive organs/tissues was performed on all control and high dose males and females, as well as on all abnormalities detected during post mortem observation. As a result of the treatment no signs of systemic toxicity were observed in the parental animals as well as in the pups. Under the study conditions, the NOEL of the substance for systemic toxicity was considered to be 1000 mg/kg bw/d (Longobardi, 2017).

Justification for classification or non-classification

Based on the results of a sub-acute oral repeated dose study with the read-across substance in rats, the substance does not warrant classification for repeated dose toxicity according to EU CLP (EC 1272/2008) criteria.