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Administrative data

Description of key information

The repeated dose toxicity of the Reaction mass of disodium N,N’-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate, has been investigated in Han Wistar (RccHan™;WIST) rats in a repeated dose, reproductive and developmental screening study conducted according to OECD TG 422. In the study, three respective groups of male and female rats (10 rats per group) were administered the substance daily (5 days/week) at concentrations of 35, 110 or 300 mg/kg bw/day by oral gavage administration for at least five weeks.  Control groups received a reverse osmosis water vehicle. On the basis that adverse effects were observed in female rats treated at 300 mg/kg bw/day, which included acute tubular necrosis in the kidneys and changes in the non-glandular stomach, the No Observed Adverse Effect Level (NOAEL) for the general systemic toxicity of the substance was determined to be 110 mg/kg bw/day.    

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422 - Combined Repeated Dose Toxicity Study and Reproductive/Developmental Toxicity Screening Study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start: 07 December 2018 Experimental Completion: 08 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Annex VIII Data Requirement
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han Wistar
Details on species / strain selection:
The Han Wistar is typically used in studies of this type, is acepted by regulatory authorities and historical control data are available.
Sex:
male/female
Details on test animals and environmental conditions:
Acclimtisation: At least 5 days prior to commencement of treatment (M/F)
Age at start of treatment: 12-13 weeks (M); 14-15 weeks (F)

Target Temperature: 20-24 degrees Centigrade
Targt Humidity: 40-70%
Light: 12 hrs light, 12 hrs dark

Bedding: Softwood based bark-free fiber, sterilized by autoclaving
Nesting material: Paper shavings
Diet: SDS VRF1 Certified, pelleted diet
Water: Potable water from the public supply via polycarbonate bottles with sipper tubes
Route of administration:
oral: gavage
Details on route of administration:
Oral gavage at a constant dose volume of 5 mL/kg
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean concentrations of the test item, in test formulations analyzed for the study were between -29% and -3.6% of nominal concentrations which were outside of the acceptability criteria of +10%/-15% from nominal on occasions. During Week 2, all results were within the acceptability criteria, confirming accuracy of the formulations. In the penultimate week, analysis of the prepared formulations for Group 2 and 4 were outside the acceptability criteria, however, in the last week of treatment, the analyzed samples met the criteria. It is considered, following investigations of the dose preparation documentation held in the raw data that the formulations were prepared correctly and therefore the animals dosed correctly. The variability, and lower than expected achieved analyzed formulation concentration values is considered to be a consequence of the physico chemical properties (i.e. high water content of 23.6%) and inherent instability of the test item within the discrete (1mL) samples supplied for analysis of achieved concentration.
Duration of treatment / exposure:
Males: 2 weeks before pairing up to necropsy after minimum of 5 weeks
Females: 2 weeks before pairing, then throughout pairing and gestation until Day 13 of lactation
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
35 mg/kg bw/day (nominal)
Dose / conc.:
110 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
No
Observations and examinations performed and frequency:
Detailed Physical Examination and Arena Observations: Pretreatment, Weekly before dosing, Days 0, 6, 13 and 20 after mating; Days 1, 6 and 12 of lactation
Sensory Reactivity and Grip Strength, Motor Activity: Week 5; Days 7-9 of lactation
Haematology and Blood Chemistry: At termination
Thyroid Hormone Analysis: At termination, Day 4 of age, Day 13 of age
Sacrifice and pathology:
METHOD OF SACRIFICE

All adult F0 animals: Carbon dioxide asphyxiation
Offspring selected for thyroid hormone sampling: Decapitation
All other offspring: Intraperitoneal injection of sodium pentobarbitone

NECROPSY

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Statistics:
For categorical data, the proportion of animals were analysed for each treated group (as appropriate) versus the control group.

For continuous data, Bartlett’s test was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups were subsequently compared with the control group, incorporating adjustment for multiple comparisons where necessary.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Administration with the test item at doses up to and including 300 mg/kg/day in males and 110 mg/kg/day in females was well tolerated. There were no significant test item-related signs observed at routine clinical examination that were considered associated with treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Group 4 was terminated early due to litter losses and poor survival prognosis of litters.

Group 4 female No. 52 was killed for welfare reasons on Day 22 of gestation. Clinical signs prior to despatch, included cold to touch, decreased activity, unresponsive, piloerection and whole-body pallor. Macroscopic examination revealed pale kidneys. Female No. 53 failed to litter and confirmed as not pregnant on Day 25 of gestation. There were no significant clinical signs. Macroscopic examination revealed stomach depressions of the non-glandular region. Group 4 female No. 54 was euthanised for reasons of animal welfare on Day 25 of gestation, due to general poor condition, the animal was suspected to have parturition problems. Group 4 female No. 56 was euthanised for reasons of animal welfare on Day 25 of gestation, due to general poor condition, the animal failed to litter. Macroscopic examination revealed pale kidneys and stomach depressions of the non-glandular region. The remining females were terminated early during lactation as it was clear that we had exceeded the maximum tolerable dose.

There were 3 deaths in the Controls. On Day 1 and 3 of lactation, control females No. 74 and 80, respectively, had total litter losses. There were no significant signs prior to their despatch. Macroscopic findings were limited to pale, inactive mammary. A third female, No. 78 was not pregnant and failed to litter. One female receiving 110 mg/kg/day was killed for welfare reasons during gestation (Day 25) as she was not pregnant and failed to litter. Macroscopic examination revealed pale and multilobular lungs.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weight gain was significantly low (Day 1 to 36), compared to Controls, in males receiving 300 mg/kg/day (69% of controls), and low in females prior to pairing (6g vs 11g in Controls)

In females receiving 300 mg/kg/day during gestation (Day 0-20), group mean body weight gain was low (77% of control). In females receiving 35 or 110 mg/kg/day, body weight was high (132 and 122% of control, respectively).

During lactation, body weight gain was significantly higher 154 and 144% of controls for females receiving 35 or 110 mg/kg/day respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was marginally but statistically significantly reduced during Days 1-15 prior to pairing for males and females receiving 300 mg/kg/day when compared with Controls. Food intake remained statistically significantly lower for females receiving test item during Day 0-20 of gestation, but a dose response was not apparent.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical investigation prior to termination revealed slightly low A/G ratios in treated males, when compared with Controls, however, in the absence of any significant findings in related parameters this finding is not considered to be of any toxicological significance.

Other changes that attained statistical significance were minor, appeared in only one sex or lacked dose relationship included slightly high alkaline phosphatase activity and triglyceride and potassium concentrations in males receiving 300 mg/kg/day and were therefore, considered to be due to normal biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
SENSORY REACTIVITY OBSERVATIONS AND GRIP STRENGTH

The sensory reactivity observations and grip strength values for males during Week 5 of treatment and females at Day 7-9 of lactation revealed no findings which were considered treatment related.

MOTOR ACTIVITY

Motor activity assessment in all groups of treated males when compared with Controls, during Week 5 of treatment revealed treatment related reductions in both high and low beam break activity. The majority of group mean high and low beam activity scores for all groups of treated males were low compared with Controls with statistical significance attained at many of the individual 6 minute time points and all total scores. Many of these scores were also below the historical control data (HCD) minimum, especially during the first half of the 1-hour recording period, generally when most exploratory behaviour would occur. All high and low beam total scores are also below the HCD minimum. The low beam total scores showed a clear dose relationship.

There was no effect of treatment on activity scores in treated females. The majority of group mean high and low beam scores for all treated females (Groups 1-3) are slightly high compared with Controls. Statistical significance was attained at two of the individual 6 minute time points for females in Group 3, however, the majority of scores are within the HCD range and therefore not considered to be related to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The analysis of organ weights after of treatment indicated, when compared with the controls, revealed high bodyweight-adjusted liver weights in treated males attaining statistical significance at 300 mg/kg/day.

Analysis of the organ weight data for females after lactation receiving 35 or 110 mg/kg/day was considered unaffected by treatment.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with Reaction mass of disodium 2,2’-{(2-hydroxy-5-nonyl-1,3-phenylene)bis[methlene(methylimino)]}diacetate and sodium [(2-hydroxy-5-nonylbenzyl)(methyl)amino]acetate were seen in the kidneys and thymus in females and the liver and thyroids of both sexes. Females given 300 mg/kg/day were euthanised early due to clinical signs and pup survival.

Kidneys: Multifocal basophilic tubules and single-cell necrosis in the tubules occurred in females given 300 mg/kg/day.
Liver: Increased glycogen was present in males and females given 300 mg/kg/day and one male given 110 mg/kg/day. Centrilobular hypertrophy was present in one male given 300 or 110 mg/kg/day.
Thyroid: Hypertrophy of follicular cells was present in males and females given 300 mg/kg/day and one male given 110 mg/kg/day.
Thymus: Involution/Atrophy was present in two females given 300 mg/kg/day.
Ovaries: Vacuolation of the corpora lutea was present in some females given 300 mg/kg/day. Similar changes were not seen in control females, but the animals were terminated at different stages of the lactation period.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 110 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
female reproductive system
Organ:
ovary
Treatment related:
not specified
Dose response relationship:
no
Relevant for humans:
yes
Conclusions:
Administration of Reaction mass of disodium 2,2’-{(2-hydroxy-5-nonyl-1,3-phenylene)bis[methlene(methylimino)]}diacetate and sodium [(2 hydroxy 5 nonylbenzyl)(methyl)amino]acetate at 300 mg/kg/day resulted in the premature death of three females with necrotic changes in the kidneys being the primary cause of death. Changes were also apparent in the stomach, thymus, bone marrow and lymph nodes. In females surviving to early termination on Day 4 of lactation, adverse changes were present in the kidneys; there were also non-adverse changes were present in the liver and thyroid glands, secondary changes were present in the thymus and ovarian changes of unknown significance was observed.

Based on the results of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 110 mg/kg/day since treatment at 300 mg/kg/day results in poor clinical condition and the welfare kill of three pregnant females with necrotic changes in the kidneys which was considered adverse.
Executive summary:

INTRODUCTION

The purpose of study was the assessment of general systemic toxic potential in Han Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration, oral by gavage, of Reaction mass of disodium 2,2'‑{(2‑hydroxy-5-nonyl-1,3-phenylene)bis[methylene(methylimino)]}diacetate and sodium [(2-hydroxy-5-nonylbenzyl)(methyl)amino]acetate, a trimerisation catalyst for industrial use, by oral gavage administration for at least five weeks.

MATERIALS AND METHODS

Three groups of ten male and ten female rats received Reaction mass of disodium 2,2'‑{(2‑hydroxy-5-nonyl-1,3-phenylene)bis[methylene(methylimino)]}diacetate and sodium [(2-hydroxy-5-nonylbenzyl)(methyl)amino]acetate at doses of 35, 110 or 300 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, throughout paring and then up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were euthanized on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, reverse osmosis water, at the same volume dose as the treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, anogenital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Blood samples were collected from selected offspring on Day 4 (not analyzed) and Day 13 of age for thyroid hormone analysis.

RESULTS

The mean concentrations of the test item, in test formulations analyzed for the study were between -29% and -3.6% of nominal concentrations which were outside of the acceptability criteriaof +10%/-15% from nominal on occasions.  During Week 2, all results were within the acceptability criteria, confirming accuracy of the formulations.  In the penultimate week, analysis of the prepared formulations for Group 2 and 4 were outside the acceptability criteria, however, in the last week of treatment, the analyzed samples met the criteria.  It is considered, following investigations of the dose preparation documentation held in the raw data that the formulations were prepared correctly and therefore the animals dosed correctly. The variability, and lower than expected achieved analyzed formulation concentration values is considered to be a consequence of the physico‑chemical properties (i.e. high water content of 23.6%) and inherent instability of the test item within the discrete (1mL) samples supplied for analysis of achieved concentration.

There were no test‑item effects related to treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

Oral administration of the test item to parental Han Wister (RccHan;WIST)rats in females at a dose level of 300 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination was not well tolerated . There were five female decedents necessitating the Group 4 females receiving 300 mg/kg/day being terminated early due to litter losses and poor survival prognosis of the litters. There were two early decedents; both of these animals receiving 300 mg/kg/day were euthanized due to welfare reasons withsigns including unresponsive, cold to touch, piloerection and pallor in one female and difficulty in parturition in another,on Days 22 and 25 of gestation, respectively, and two females failed to litter. Furthermore, one female receiving 300 mg/kg/day was prematurely euthanized as a result of total litter loss. In addition, two Control females were euthanized due to premature litter loss on Days 1 and 3 of lactation, respectively.

There were no test-item related signs observed during the detailed physical examination and arena observations, no post-dosing observations and no effects on sensory reactivity, grip strength or adverse effects on motor activity.

Overall body weight gain,when compared with controls, was statistically significantly(Days 1 to 36) in males receiving 300 mg/kg/day and low in females prior to pairing. In females receiving 300 mg/kg/day from Days 0-20 of gestation, overall body weight gain was low. In females receiving 35 or 110 mg/kg/day, body weight gain was high. During lactation, body weight gain wasstatistically significantly higherfor females receiving 35 or 110 mg/kg/day.

Food intake was marginally reduced,when compared with controls,during Days 1-15 prior to pairing for males and females receiving 300 mg/kg/day. Food intake remained statistically significantly lower for females receiving 300 mg/kg/day test item during Days 0‑20 of gestation. Food intake was unaffected by treatment at dose levels of 35 or 110 mg/kg/day.

There was notest-item relatedeffect on estrous cycles or pre-coital interval. The rate of conception and fertility index were unaffected by treatment. The number of copulation plugs observed in the cages of animals that received 300 mg/kg/day tended to be lower than that of Controls. There was notest-item relatedeffect on sperm count estimates and at termination all females were in diestrus. A shift in gestation length was apparent for females receiving 300 mg/kg/day;it was slightly longer in one female exhibiting a 25.5-day gestation length, and five females exhibited a 23.5 day gestation length, compared with 23 days commonly seen in the controls. The gestational length of females receiving 35 or 110 mg/kg/day was unaffected by treatment.

There were no test-item related effects in hematological and biochemical investigations prior to termination.

Organ weight changes were limited to increasedbodyweight adjusted liver weights, when compared with controls, in males administered 110 or 300 mg/kg/day and was not considered adverse. 

The macroscopic examination performed after a minimum of 5 weeks of treatment revealed no test item related lesions.

Histopathological evaluation of retained tissues revealed treatment related changes in the kidneys, liver, thyroid glands and thymus.

At 300 mg/kg/day offspring body weight on Day 1 of age were low (mean 5.5g and 5.2g in males and females, respectively), when compared to controls (6.4g and 6.2g in males and females, respectively) and body weight gain was markedly low (gains of 0.1 to 1.1g, compared to 3.5 or 3.3g in control males and females, respectively) in 2 out of 4 litters during Day 1‑4 of age. Mean offspring bodyweight and mean bodyweight gain for male and female offspring of parents treated with test item at 30 or 110 mg/kg/day were not affected by treatment when compared with Controls.

There were no clinical signs in the offspring considered to be related to treatment with the test item. Litter size up to Day 13 of age was unaffected by treatment in females receiving 35 or 110 mg/kg/day. Litter size at 300 mg/kg/day was not assessed as these were terminated early. Group mean percentage of post implantation survival, live birth, the viability and lactation indices for females receiving 35 or 110 mg/kg/day were not affected by treatment. There were no effects on sex ratio that were associated to treatment with test item. There was no effect of parental treatment on the anogenital distance of male or female offspring on Day 1 of age. There were no macroscopic findings which were considered related to parental treatment with the test item.

CONCLUSION

Administration of Reaction mass of disodium 2,2’-{(2-hydroxy-5-nonyl-1,3-phenylene)bis[methlene(methylimino)]}diacetate and sodium [(2‑hydroxy‑5‑nonylbenzyl)(methyl)amino]acetate at 300 mg/kg/day resulted in the premature death of three females with necrotic changes in the kidneys being the primary cause of death. Changes were also apparent in the stomach, thymus, bone marrow and lymph nodes. In females surviving to early termination on Day 4 of lactation, adverse changes were present in the kidneys; there were also non-adverse changes were present in the liver and thyroid glands, secondary changes were present in the thymus and ovarian changes of unknown significance was observed.

Based on the results of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 110 mg/kg/day since treatment at 300 mg/kg/day results in poor clinical condition and the welfare kill of three pregnant females with necrotic changes in the kidneys which was considered adverse.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
110 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study conducted according to GLP and OECD Test Guidelines
System:
other:
Organ:
kidney
stomach

Additional information

The repeated dose toxicity of the substance, has been investigated in Han Wistar (RccHan™;WIST) rats in a repeated dose, reproductive and developmental screening study conducted according to OECD TG 422.  The study included an assessment of endpoints relevant to endocrine disruption properties.  In the study, three respective groups of male and female rats (10 rats per group) were administered the substance daily (5 days/week) at concentrations of 35, 110 or 300 mg/kg bw/day by oral gavage administration for at least five weeks.  Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks.  Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation.  Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation.  Control male and female groups received the vehicle, reverse osmosis water, at the same volume dose as treated groups.

Investigations of general systemic toxicity included: clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, organ weight and macroscopic pathology and histopathology.

The dose level of 300 mg/kg bw/day was not well tolerated in parental female rats during the two weeks prior to pairing, during pairing and then up to termination and a number of female decedents on this study that necessitated the Group 4 females receiving 300 mg/kg/day being terminated early due to litter losses and poor survival prognosis of the litters.  There were two early decedents, both animals were receiving 300 mg/kg/day and were killed due to welfare reasons on Day 22 and Day 25 of gestation, respectively and two females failed to litter.  Furthermore, one female receiving 300 mg/kg/day were prematurely killed as a result of a total litter loss.  In addition, two Control females were killed prematurely litter loss on Day 1 and 3 of lactation.

No treatment-related signs were observed during detailed physical examination and arena observations, there were no post-dosing observations and no effects on sensory reactivity, grip strength or adverse effects on motor activity were noted. Overall, body weight gain was significantly low in males receiving 300 mg/kg/day (days 1 to 36), and low in females prior to pairing and receiving 300 mg/kg/day during gestation (Day 0-20). In females receiving 35 or 110 mg/kg/day, body weight gain was high and was significantly higher during lactation.  In the 300 mg/kg bw/day dose group, food intake was marginally reduced in males and females during days 1-15 prior to pairing and remained statistically significantly lower for females during day 0 to 20 of gestation.  Food intake during lactation was unaffected by treatment at 35 or 110 mg/kg/day.

Haematological and biochemical investigations prior to termination were unaffected by treatment. Changes in organ weight were limited to high bodyweight adjusted liver weights in males treated at 110 or 300 mg/kg/day. No treatment-related lesions were reported in macroscopic examinations.

Histopathological evaluation of retained tissues revealed treatment related changes in the kidneys, liver, thyroid glands and thymus.  In the kidneys, multifocal basophilic tubules and single-cell necrosis in the tubules occurred in females treated at 300 mg/kg/day.  Increased glycogen in the liver was present in males and females given 300 mg/kg/day and one male given 110 mg/kg/day.  Centrilobular hepatocyte hypertrophy was present in one male given 300 or 110 mg/kg/day.  Hypertrophy of follicular cells of the thyroid gland was present in males and females given 300 mg/kg/day and one male given 110 mg/kg/day and involution/atrophy was present in the thymes of two females given 300 mg/kg/day.

Administration of the substance at 300 mg/kg/day resulted in the premature death of three females with necrotic changes in the kidneys being the primary cause of death.  Changes were also apparent in the stomach, thymus, bone marrow and lymph nodes.  In females surviving to early termination on Day 4 of lactation, adverse changes were present in the kidneys, non-adverse changes in the liver and thyroid glands, secondary changes in the thymus and ovarian changes of unknown significance.

Based on the results of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 110 mg/kg/day due to the adverse changes in the kidneys of females receiving 300 mg/kg/day.  

Justification for classification or non-classification

Based on the findings of this study, no classification is required.