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Description of key information

NOAEL (oral; sub-chronic; rat) = 500 mg/kg bw/day


NOAEC (inhalation; chronic; rat) = 0.13 mg/L air (corresponding to 1000 ppm, analytical concentration)


NOAEL (dermal; sub-acute; rat) = 20 mL/kg bw/day

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
0.13 mg/m³
Study duration:
chronic
Experimental exposure time per week (hours/week):
30
Species:
rat

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

The Repeated dose toxicity via the oral route is predominantly evaluated considering data on the substance itself, from a sub-chronic (90 days) study on rats. Further studies on similar substances are used as supporting.


No experimental data on the repeated dose toxicity of the substance was available for the inhalation and dermal route, therefore, repeated dose toxicity of the substance was evaluated using a variety of experimental studies performed on the structural analogues of the substance. Justification for the use of a read-across approach is detailed in Section 13 of this Registration Dossier.


 


Repeated Dose Toxicity: Oral


The key study was conducted to evaluate the potential toxicity of dibutyl phosphonate in Wistar rats following daily oral gavage administration for 90 consecutive days according to the OECD 408 and under GLP conditions. Four groups of 10 female and 10 male rats each were exposed at 0, 100, 250 and 500 mg/kg bw/day for 90 days. A recovery period of 28 days for the vehicle control and the highest groups also run. The vehicle chosen was corn oil. 


Dose formulation analysis was performed in order to evaluate the homogenicity and the active ingredient concentration. The formulations were prepared every day and were administred shortly after their preparation. During the experimental period, all rats were observed for signs of toxicity, morbidity, and mortality. Body weight was recorded at weekly intervals. Body weight change and food consumption were calculated at weekly intervals. An ophthalmological examination was performed before treatment and sacrifices. Neurobehavioral observations were performed at weekly intervals. Functional observational battery was performed during 12th week in main groups and during 4th week in recovery groups. At the end of the treatment and recovery periods, clinical pathology estimations were performed in all rats. Vaginal smears was examined on the day of necropsy in all female rats. At the end of the treatment and recovery periods, all rats were sacrificed and subjected to gross pathological examination. The weight of selected organs was recorded for each rat. Microscopic examination of tissues and organs was performed in vehicle control (G1) and high dose (G4) groups. Histopathological examination revealed treatment related alterations in non-glandular stomach. Therefore, stomach was processed from low, mid, and recovery groups of both sexes.


Results


Mortality and morbidity: No mortality or morbidity was observed during the treatment and recovery periods.


Clinical Observations: All rats were normal, throughout the dosing period, in control, low, and mid dose groups. Mild salivation was observed during post dosing observation in male and female rats of high dose treated group. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity (Wook-Joon Yu at al, 2011). Therefore, it was considered that the transient salivation observed in this study was of doubtful toxicological significance, since no related changes were observed in the low and mid dose groups.


Ophthalmological examination: Ophthalmological examination of all rats did not reveal any abnormality.


Neurobehavioural Observations: No treatment-related change was observed in neurobehavioural observations performed in male and female rats from treatment groups.


Functional Observational Battery: No treatment-related change was observed in functional observational battery parameters performed in male and female rats from treatment groups


Body Weight and Body Weight Change: No treatment-related change was observed in body weight and percent body weight change in rats from treatment groups when compared with those of respective vehicle control groups.


Food Consumption: Intermittent reduction in the mean food consumption was observed in male and female rats of treatment groups. Also, body weight growth of these animals were not that much impacted. These changes could be considered due to irritant properties of test item in gastric mucosa and were marginal. Hence, it could be considered as effect of the test item treatment but not adverse in nature.


Clinical pathology: No treatment-related change was observed in haematology, coagulation, clinical chemistry, and urinalysis parameters of rats from treatment groups when compared with those of respective vehicle control groups.


Organ weight: No treatment-related change was observed in the terminal body weight, organ weight, and relative organ weight of rats from treatment groups when compared with those of respective vehicle control groups.


Macroscopic examination: Internal gross examination revealed increase in thickness of non-glandular stomach in all 3 treated main groups of both sexes (except in G2 males) in dose dependent manner. Lesion was considered as related to the test item treatment. The change was considered as insignificant due to lack of human relevance as non-glandular stomach is absent in humans.


Microscopic examination: Microscopic examination revealed hyperkeratosis and mucosal hypertrophy/hyperplasia in non-glandular stomach in all treated main groups of both sexes in dose dependent manner. Lesions were related to the test item treatment and considered as insignificant due to lack of human relevance as non-glandular stomach is absent in humans (Jeff Mckee, 2011). Marked recovery was observed in the effects after the recovery period.


Conclusion:


There was no treatment-related change observed in in-life phase parameters except the clinical signs like salivation in male and female rats treated with 500 mg/kg b. wt. and intermittent decrease in food consumption in male and female rats treated with 100, 250 and 500 mg/kg b. wt. These changes were not having toxicological significance in this study and considered as non-adverse in nature.


Haematology, coagulation, urine analysis and organ weights parameters did not reveal any test item treatment related change.


On gross examination, treatment revealed thickness of non-glandular stomach in all treatment groups, which was associated with histopathological lesions. The change was considered as insignificant due to lack of human relevance as non-glandular stomach is absent in humans.


Hyperkeratosis and mucosal hypertrophy/hyperplasia in non-glandular stomach was observed in all treated main groups of both sexes in dose dependent manner and considered as insignificant due to lack of human relevance as non-glandular stomach is absent in humans.


Therefore, the No Observed Adverse Effect Level (NOAEL) of dibutyl phosphonate is 500 mg/kg b. wt./day.


Several relevant supporting studies were considered in order to assess the oral repeated dose toxicity. The most relevant of these are two GLP, OECD studies on rats performed on the two primary metabolites of the substance. The first of these is a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, performed on SC11 according to the OECD Guideline 422. This study provided a sub-acute NOAEL of 250 mg/kg bw/day among rats. The second of these studies was performed on SC09 and according to a method similar to the OECD Guideline 408. This study provided a sub-chronic NOAEL of 125 mg/kg bw/day among rats. Rapid and complete hydrolysis of the substance occurs in aqueous medium, rendering both SC09 and SC11, therefore, data on these two substances is sufficient to evaluate this endpoint. Based on these values, the NOAEL for the substance can be considered the lower of these two chemicals: 125 mg/kg bw/day.


An increasing trend in toxicity was observed with decreasing ester length. Specifically, the structural analogues with methanol, ethanol and octanol esters in place of the butanol esters rendered NOAEL values of 50, 30-150 and 300 mg/kg/d, respectively, in line with the predicted NOAEL range of 125-250 mg/kg bw/day for the substance. This was further supported by a battery of tests on rats performed by the National Toxicology Program, but without GLP or an OECD guideline, provided a sub-acute NOAEL of 250 mg/kg bw/day and a sub-chronic NOAEL of 50 mg/kg bw/day (females) and 100 mg/kg bw/day (males).


Based on this supporting evidence, a sub-chronic NOAEL value of 125 mg/kg bw/day is accepted for use of this substance.


Repeated Dose Toxicity: Inhalation


Few inhalation repeated dose toxicity studies performed on structural analogues were available. Of these, a study on a primary metabolite of the substance (SC09) was awarded a reliability value of 4, and is therefore not considered in the evaluation of inhalation repeated dose toxicity. Two experimental studies performed on rats and according to methods equivalent/similar to OECD guidelines were identified, both performed on the more sensitive structural analogue with methanol esters in place of the butanol esters. One of these studies is a sub-acute study (OECD Guideline 412) and the other is a chronic study (OECD Guideline 453); the resulting NOAEL values are 6.63 mg/L (equivalent to 5010 ppm, analytical measurement) and 0.13 mg/L (equivalent to 100 ppm), respectively. The only other inhalation study considered for the evaluation of repeated dose toxicity was performed on a test animal not usually used (monkey) and not according to an OECD Guideline, and was therefore not considered in the calculation of NOAEL for the substance.


Due to the wide range of values obtained for the repeated dose toxicity of the substance via inhalation, the more conservative chronic NOAEC value of 0.13 mg/L is applied in order to consider a worst-case scenario approach. In any case, it should be noted that this value was not used in the DNEL calculation as it is considered too conservative and not representative of the true toxicity of the substance.


Repeated Dose Toxicity: Dermal


One experimental study on a structural analogue was identified in order to assess the dermal repeated dose toxicity of the substance. A sub-chronic NOAEL value of 20 mL/kg bw/day was set in this study. However it should be noted that this study was perfored (i) not according to principles of GLP, (ii) not according to an OECD Guideline, (iii) on rabbits, (iv) with poor documentation, (v) as a limit test, and (vi) on a primary metabolite of the substance. Therefore, caution should be exercised in the application of a NOAEL for dermal repeated dose toxicity using this study.

Justification for classification or non-classification

According to the CLP Regulation (EC) No. 1272/2008, substances are classified as specific target organ toxicants following repeated exposure, and are placed in one of two categories, depending on the nature and severity of the effect(s) observed. Substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure, are classified in Category 1 for target organ toxicity (repeat exposure). Classification in Category 1 is applicable when significant toxic effects are observed in a 90-day repeated-dose animal study at low concentrations (< 10 mg/kg bw/day in oral studies, and < 20 mg/kg bw/day in dermal studies) (CLP Regulation (EC) No. 1272/2008: Annex 1, Part 3, Table 3.9.2).


Substances which can be presumed to have the potential to be harmful to human health following repeated exposure, based on evidence from animal studies, are classified in Category 2. Classification in Category 2 is applicable when significant toxic effects are observed in a 90-day repeated-dose animal study at generally moderate exposure concentrations (10 to 100 mg/kg bw/day in oral studies, and 20 to 200 mg/kg bw/day in dermal studies) (CLP Regulation (EC) No. 1272/2008: Annex 1, Part 3, Table 3.9.3).


 


Based on the sub-chronic, oral NOAEL value of 500 mg/kg bw/day in rats, no classification of the test item is warranted for specific target organ toxicity – repeated exposure according to the CLP Regulation (EC) No. 1272/2008.

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