Dilithium tetraborate

Endpoint summary

Description of key information

In a combined 28 -day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422) of dilithium tetraborate by oral gavage in rats using doses 0,15, 50 and 150 mg/kg/day, no treatment-related general systemic changes were noted. The parental NOAEL (male and female) based on the outcome of this study is equal to or greater than 150 mg/kg (bw).

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 TG 422

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

2015

Deviations:

yes

Remarks:

However, none of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

At initiation of dosing, males were 10 weeks old and weighed between 273 and 309 g and females were 13 weeks old and weighed between 198 and 233 g.The animals were allowed to acclimate to the Test Facility toxicology accommodation for 8 days prior to start of the pre-test period (females) or 8 days before the commencement of dosing (males).
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 22°C with an actual daily mean relative humidity of 42 to 73%. A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Pelleted rodent diet was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage

Vehicle:

other: MOL WO M 46 medicinal white oil

Details on oral exposure:

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item.Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle.

Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during dose administration. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 29 days. Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 38-46 days.

Frequency of treatment:

Once daily, 7 days/week

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels in this study were selected to be 0, 15, 50 and 150 mg/kg/day, based on the results of the dose range finder. Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- (F0-Generation ). Time schedule: On treatment Days 1-15, these clinical observations were at least conducted 1 hour ± 15 minutes after dosing. From Day 16 of treatment onwards, these clinical observations were conducted during the intervals 0-15 minutes and 1 hour ± 15 minutes after dosing

BODY WEIGHT: Yes
- (F0-Generation ). Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
A terminal weight was recorded on the day of scheduled necropsy (fasted for males, non-fasted for females).

FOOD CONSUMPTION: Yes
-Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottle.

FUNCTIONAL TESTS:
-Functional tests were performed on the selected 5 males during Week 4 of treatment and on 5 selected females during the last week of lactation (i.e. PND 6-13 for Groups 1-3 and PND 2-6 for Group 4) or post-coitum Day 23-26 (Group 4). These tests were started at least 1 hour ± 15 minutes after dosing, after completion of clinical observations.

Sacrifice and pathology:

- Male animals: Which sired and failed to sire. Following completion of the mating period (a minimum of 28 days of administration). All males surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy. Water was available.
- Maternal animals: Females which delivered (PND 14-16) and females which failed to deliver (with evidence of mating, post-coitum day 25-27. Dams with no surviving pups were euthanized within 24 hours after the last pup is found dead or missing

Other examinations:

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs ( See Table 1,2,3,)

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings were noted only in females at 150 mg/kg and consisted of piloerection, mostly on one or a few days at the beginning of the lactation period, and, less frequently, hunched posture on a few days during gestation or lactation. The piloerection noted incidentally in a few females at 50 mg/kg and piloerection and hunched posture at 150 mg/kg was considered not to reflect an adverse effect on general health.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No animals died spontaneously or were euthanized for humane reasons, except for one female at 150 mg/kg (no. 73) which was euthanized due to delivery difficulties.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight gain was observed in three 150 mg/kg females between Days 17-20 of gestation and during lactation. For two females the lower weight gain at the end of gestation was related to a low number of growing fetuses (one or none). The reduced weight gain during lactation was accompanied by a marked decrease in food consumption (two of the three lactating females at 150 mg/kg had total litter loss at PND 4 or 6).

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related, non-adverse hematological changes were observed at 50 mg/kg (females only) and 150 mg/kg (both sexes). The changes at 150 mg/kg consisted of higher numbers of reticulocytes and lower hemoglobin concentration in both sexes, and increased number of platelets, higher red blood cell distribution width (RDW) and lower hematocrit and mean corpuscular hemoglobin (MCH) in females. Altered red blood cell values in 150 mg/kg females mostly occurred in the females that had a different physiological status (i.e. no healthy offspring) and shorter study duration than controls. Females at 50 mg/kg had lower values for hemoglobin, hematocrit and numbers of white blood cells (total, lymphocytes and neutrophils).
The red blood cell changes at 150 mg/kg were associated with extramedullary hematopoieses in the spleen in both sexes and increased spleen weights and macroscopic enlargement/thickening of the spleen in females. A slight increase in splenic extramedullary hematopoiesis noted in 50 mg/kg males occurred in the absence of changes in red blood cell parameters. The extramedullary hematopoiesis in the spleen was not accompanied by any degenerative changes and, therefore, regarded as non-adverse.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Non-adverse clinical chemistry changes were observed at 150 mg/kg. Cholesterol was decreased in both sexes, other changes generally occurred in only one sex (lower sodium and higher total bilirubin and bile acids in males; lower urea and higher albumin and calcium in females) or the direction of the changes was different in the two sexes (alanine aminotransferase (ALAT), total protein, potassium). The changes in 150 mg/kg females mostly occurred in the females that had a different physiological status and study duration than controls (exceptions: lower cholesterol and ALAT). The changes were regarded as non-adverse as they were not associated with adverse anatomic pathology findings and remained within physiological ranges.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher absolute and/or relative weights of the thyroid and adrenal glands were noted in females at 150 mg/kg. In the absence of corroborative microscopic findings these organ weight changes were regarded as non-adverse.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic findings were noted in females treated at 150 mg/kg and consisted of an enlarged spleen in 2/10 females and a thickened spleen in one of these two females (microscopic correlate: extramedullary hematopoiesis in the spleen). The extramedullary hematopoiesis in the spleen was not accompanied by any degenerative changes and, therefore, regarded as non-adverse. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased extramedullary hematopoiesis was present in males starting at 50 mg/kg and in females at 150 mg/kg (See above - not considered to be adverse) .Inflammatory cell infiltrate and necrosis of the heart was present in a single female (no. 75) at 150 mg/kg, which as only ocurring in one test animal is unlikely to be a treatment related effect.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects reported in the organ systems

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

No general systemic treatment related effects for males or females were reported in a 28 -day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422) of dilithium tetraborate by oral gavage in rats. The NOAEL (male and female) is equal or greater than 150 mg/kg(bw).

Executive summary:

In a combined 28 -day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422) of dilithium tetraborate by oral gavage in rats using doses 0,15, 50 and 150 mg/kg/day, no treatment-related general systemic changes were noted. The parental NOAEL (male and female) based on the outcome of this study is equal to or greater than 150 mg/kg (bw).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 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

Justification for classification or non-classification

In a combined 28 -day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422) of dilithium tetraborate by oral gavage in rats using doses 0,15, 50 and 150 mg/kg/day, no repeated dose adverse effects were reported. Therefore the classification criteria (STOT-RE) as according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) / Regulation (EC) No 1272/2008 (including all amendments) are not fulfilled.