Ethyl 3-[[bis(1-methylethoxy)phosphinothioyl]thio]propionate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effects on reproductive organs were observed in repeated dose toxicitxy studies (see 7.5.1).

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No adverse effects on reproductive organs were observed in a subacute oral toxicity study and ina subchronic oral toxicity study with a structural analogue (see 7.5.1). Since histopathology of reproducitve organs has proven to be a very sensitive marker of reproductive function, these data is considered adequate information at the current tonnage level.

Effects on developmental toxicity

Description of key information

A study for developmental toxicity and teratogenicity has been performed with the read across substance in rats following OECD testing guideline 414 and GLP (BASF 2013) at 50, 150 and 450 mg/kg bw. There were no indications of fetotoxicity, embryotoxicity or teratogenicity.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Aug 2012 - 20 Feb 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: About 11-13 weeks
- Weight at study initiation: 171g
- Housing: single caging
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-08-13 To: 2012-08-29

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed by shaking until it was dissolved.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test item is miscible with corn oil. It is not soluble in water.
- Concentration in vehicle: 1250 - 10000 mg/100 mL
- Amount of vehicle (if gavage): 4 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC-method

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

gestation days 6- 19

Frequency of treatment:

daily

Duration of test:

14 days

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

400 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on range-finder study with pregnant rats and doses of 250, 500 and 1000 mg/kg bw (project no. 01 R0415/11 R043, non GLP).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Morbidity, pertinent behavioral changes and/or signs of overt toxicity. were checked twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GO 0 to 20).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GO 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.


POST-MORTEM EXAMINATIONS: No
- Sacrifice on gestation day: GD 20

Other: Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.

Haematology parameters:
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes

Clinical chemistry
1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Serum y-glutamyl transferase
5. lnorg. phosphate
6. Calcium
7. Urea
8. Creatinine
9. Glucose
10. Total bilirubin
11. Total protein
12. Albumin
13. Globulins
14. Triglycerides
15. Cholesterol

Organ weights were determined for adrenal glands, liver and kidneys. These organs were also embededd for potential histopathology investigation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Site of implantations in the uterus

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter ]

Statistics:

DUNNETT's test: Food consumption, body weight, body weight change, DUNNETT's test
corrected body weight gain, carcass weight, weight of
the unopened uterus, weight of the placentas and
fetuses, corpora lutea, implantations, pre- and
postimplantation losses, resorptions and live fetuses

FISHER's exact test
Number of pregnant animals at the end of the study, FISHER's exact test mortality rate (of the dams) and number of litters with fetal findings

WILCOXON test
Proportion of fetuses with findings per litter

KRUSKAL-WALLIS and WILCOXON test
Clinical pathology parameters
Absolute and re lative organ weights

Indices:

sex ratio

Historical control data:

Included in the final report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All 25 females of the high-dose group (400 mg/kg bw/d) and 7 females of the mid-dose group (150 mg/kg bw/d) showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 10 minutes) and was initially observed on GD 7. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 50 mg/kg bw/d during the entire study period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the average body weight gain of the low-, mid- and high-dose groups (50, 150 or 400 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period. However, there were some statistically significantly increased mean body weight gain values: in test groups 1 and 2 during GD 8-10 and in test group 3 during GD 17-19 and, calculated for the treatment period, GD 6-19. They were considered spontaneous events.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) of test groups 1-3 (50, 150 or 400 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group.
The statistically significantly increased corrected body weight gain in test group 3 was considered as a spontaneous change. These values reflect the normal range of biological variation inherent in the strain of rats used for this study and were not considered to be substance-induced.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of the dams in test groups 1-3 (50, 150 or 400 mg/kg bw/d) was generally comparable to the concurrent control group throughout the whole study period. This includes the statistically significantly increased food consumption values in test group 3 (400 mg/kg bw/d) on GD 15-17 and in test group 2 (150 mg/kg bw/d) on GD 19-20.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
In dams of test group 3 (400 mg/kg bw/d), red blood cell (RBC) counts, hemoglobin, hematokrit and mean corpuscular volume (MCV) values were lower compared to controls. However, all mentioned parameters were within historical control ranges.
In dams of test group 2 (150 mg/kg bw/d), white blood cell (WBC) counts as well as absolute neutrophil and lymphocyte counts were increased, but the alterations were not dose-dependent. In females of test group 3 (400 mg/kg bw/d), absolute lymphocyte counts were still higher compared to controls, but the mean was within the historical control range.
Therefore, all mentioned hematology parameter changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In dams of test groups 2 and 3 (150 and 400 mg/kg bw/d), alanine aminotransferase (ALT) activities, urea and inorganic phosphate levels were increased, whereas total protein, albumin, globulin, cholesterol and calcium levels were decreased. Apart from total protein, globulin and cholesterol values in dams of test group 3 (400 mg/kg bw/d), all other mentioned levels were within historical control ranges.
Additionally, in dams of test group 3 (400 mg/kg bw/d) alkaline phosphatase (ALP) activities and glucose levels were increased whereas creatinine levels were decreased. However, also these values were within historical control ranges. Therefore, apart from decreased total protein, globulin and cholesterol levels in individuals of test group 3 (400 mg/kg bw/d) all other mentioned alterations were regarded as incidental and not treatment-related.
Finally, in dams of test groups 2 and 3 (150 and 400 mg/kg bw/d) total bilirubin levels were lower compared to controls. A decrease of total bilirubin can be due to a hypoproliferative anemia which was not present in these animals. Most probably, lower bilirubin levels were based on a greater conjugation rate because of liver enzyme induction followed by an increased excretion of bilirubin via bile. This effect was regarded as adaptive and not adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The mean gravid uterus weights of the animals of test groups 1-3 (50, 150 and 400 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
A dose related increase of the absolute and relative organ weights of the adrenals and liver was recorded for test groups 2 and 3 (150 and 400 mg/kg bw/d). The apparently increased absolute kidney weights were not related to dose and no significant change was seen for the relative weights. Therefore they were not considered treatmentrelated.

Gross pathological findings:

no effects observed

Description (incidence and severity):

During necropsy no test substance-related gross lesions were noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 150 and 400 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The mean placental weights of the low-, mid- and high-dose groups (50, 150 and 400 mg/kg bw/d) were comparable to the corresponding control group.

Key result

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The mean fetal weights of test groups 1, 2 and 3 (50, 150 and 400 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (50, 150 and 400 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

Description (incidence and severity):

External malformations were recorded for one mid-dose fetus (150 mg/kg bw/d) and one high-dose fetus (400 mg/kg bw/d). Both fetuses had more than one malformation affecting either the head or the abdomen. The total incidence of external malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.
No external variations were recorded.
One unclassified external observation, i.e. placentas fused, was recorded in one fetus of the control and the low-dose group (50 mg/kg bw/d), respectively. This finding was not considered biologically relevant. It can be found in the historical control data at higher incidences.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal malformations were recorded for two low-dose fetuses in two litters (50 mg/kg bw/d). Another malformation in a mid-dose fetus (150 mg/kg bw/d) was part of a multiple malformation in this offspring. The total incidence of skeletal malformations in treated animals did not differ significantly from the control group.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing. However, the incidence of bipartite processus xiphoideus was statistically significantly increased in test group 2 (150 mg/kg bw/d) and, as a consequence of this occasional increase, also the incidence of total fetal skeletal unclassified cartilage observations in this test group. However, this finding showed no dose-dependency and was therefore assessed to be without biological relevance.

Visceral malformations:

no effects observed

Description (incidence and severity):

One soft tissue malformation, which was part of a multiple malformation, was observed in one fetus of the high-dose group (400 mg/kg bw/d). The overall incidences of soft tissue malformations were comparable to those found in the historical control data.
Three soft tissue variations, i.e. short innominate, dilated renal pelvis and dilated ureter, were detected in all test groups including the control (test groups 0-3; 0, 50, 150 and 400 mg/kg bw/d). The incidences of these variations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant. They can be found in the historical control data at higher incidences.
Two unclassified soft tissue observation, i.e. discolored vitreous humor and discolored spleen, were recorded in one fetus each of test group 2 (150 mg/kg bw/d) and test group 3 (400 mg/kg bw/d). These findings were not considered biologically relevant.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Other embroytoxic/fetal parameters (implantation losses, sex ratio, weights, etc) were normal. Details are presented in table 6.

Key result

Dose descriptor:

NOEL

Effect level:

>= 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Absolute organ weights of dams
	control	50 mg/kg bw	150 mg/kg bw	400 mg/kg bw
adrenal gland	67.84	68.25	76.12**	80.8**
SD	9.48	9.88	10.39	12.16
kidney	1.56	1.67	1.67	1.7
SD	0.12	0.18	0.17	0.14
liver	11.22	11.57	11.99**	14.55**
SD	1.85	1.3	0.934	1.18

*: p <= 0.05, **: p <= 0.01

Table 2: Terminal body weight (dams)
	control	50 mg/kg bw	150 mg/kg bw	400 mg/kg bw
body weight	241	246	247	250
SD	13	19	16	14

Table 3: Relative organ weights of dams
	control	50 mg/kg bw	150 mg/kg bw	400 mg/kg bw
adrenal gland	0.028	0.028	0.031*	0.032*
SD	0.004	0.004	0.004	0.004
kidney	0.648	0.68	0.679	0.679
SD	0.043	0.048	0.052	0.043
liver	4.65	4.7	4.86**	5.82*
SD	0.36	0.25	0.28	0.44

*: p <= 0.05, **: p <= 0.01

Table 4: Summary of all classified fetal skeletal observations

		control	50 mg/kg bw	150 mg/kg bw	400 mg/kg bw
litters evaluated		25	24	25	25
fetuses evaluated		124	123	126	121
	fetuses live	124	123	126	121
	fetuses dead	0	0	0	0
Total malformations
	fetal incidence; N	0	2	1	0
	litter incidence, N	0	2	1	0
	affected fetuses/litter Mean %	0	1.4	0.8	0
Total variations
	fetal incidence; N	122	121	125	121
	litter incidence, N	25	24	25	25
	affected fetuses/litter Mean %	98.4	98.6	99.2	100

Table 5: Summary of all classified fetal soft tissue observations

		control	50 mg/kg bw	150 mg/kg bw	400 mg/kg bw
litters evaluated		25	24	25	24
fetuses evaluated		112	111	109	108
	fetuses live	112	111	109	108
	fetuses dead	0	0	0	0
Total malformations
	fetal incidence; N	0	0	0	1
	litter incidence, N	0	0	0	1
	affected fetuses/litter Mean %	0	0	0	0.8
Total variations
	fetal incidence; N	4	4	3	7
	litter incidence, N	4	3	3	5
	affected fetuses/litter Mean %	3.6	3.3	2.5	5.8

Table 6: Summary of all classified fetal external observations

		control	50 mg/kg bw	150 mg/kg bw	400 mg/kg bw
litters evaluated		25	24	25	24
fetuses evaluated		236	234	235	229
	fetuses live	236	234	235	229
	fetuses dead	0	0	0	0
Total malformations
	fetal incidence; N	0	0	1	1
	litter incidence, N	0	0	1	1
	affected fetuses/litter Mean %	0	0	0.4	0.4

Table 7: Overview of reproductive data

	control	50 mg/kg bw	150 mg/kg bw	400 mg/kg bw
postimplantation loss %	6%	5.20%	6.20%	7.30%
preimplantation loss %	8.8	6.4	5.1	7.1
Total resorptions	0.6	0.5	0.6	0.6
live fetuses/per dam	9.4	9.8	9.4	9.2
placental weights, mean, g	0.48	0.49	0.48	0.56
fetal weights, mean, g	3.6	3.7	3.6	3.6
corpus lutea, mean	11	11	10.5	10.5
Dam uterine weight, mean	52.6g	54.9g	52.4g	51.9g
Corrected body weight gain	38.9g	42.1g	42.9g	47g*
Pregnant at terminal sacrifice	25	24	25	25
conception rate, %	100	96.4	100	100
% live female fetuses	52.5	48.7	50.6	53.7
% live male fetuses	47.5	51.3	49.4	46.3

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 400 mg/kg bw/d caused evidence of maternal toxicity, such as dysregulated liver cell metabolism and dose-relatedly increased liver and adrenal weights. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 150 mg/kg bw/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 400 mg/kg bw/d. There were no toxicologically relevant adverse fetal findings evident.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A GLP-compliant study following OECD testing guideline 414 has been performed in rats with the read across substance (see attached document for read across justification). The test item was administered by gavage to groups of 25 pregnant rats using corn oil as a vehicle. Based on the results of a dose-range-finding study in pregnant rats, dose levels of 50, 150 and 400 mg/kg bw were chosen for the main study. Administration of the test article was performed on gestation days 6 through 19. The control group was dosed with the vehicle alone. Extra investigations on known target organs (liver, adrenal glands and kidneys) were included in the study design. On GD 20, all females were sacrificed and examined, including weight determinations of the unopened uterus and the placentas. For each dam, corpora lutea were counted and number and distribution of implantation sites were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal findings.

No adverse effects on fetal or embryo development and no teratogenicity were observed. Dams were affected by treatment as indicated by an absolute and relative increase in the weights of liver and adrenal glands at 150 and 400 mg/kg bw. At 400 mg/kg bw, mild changes were observed in clinical chemistry (decrease in cholesterol, bilirubin and total protein). This could indicate induction of xenobiotic liver metabolism. Dam body weight development was not affected, nor were there signs of morbidity.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for toxicity to reproduction is not warranted under Regulation (EC) No.1272/2008.