Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides, reaction products with polyethylenepolyamines and tall-oil fatty acids, humates hydrochlorides

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-03-18 - 2019-11-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

yes

Remarks:

thyroid gland not weighed and histopathological examined; anogenital distance not measured in live fetuses

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Substance type: UVCB
- Physical state: powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: between 67 and 76 days old
- Weight at study initiation: 209 - 300 g (range)
- Fasting period before study: no
- Housing: 2 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 to 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 21
- Humidity (%): 38 - 73
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 Mar 2019 To: 11 Apr 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing formulations were prepared and dispensed daily. Formulations were delivered to the animal unit immediately. The test and control items were administered to the appropriate animals by once daily oral gavage from Gestation Day 6 to 20 (where Gestation Day 0 was the day of detection of vaginal plug). The volume for each animal was based on the most recent body weight measurement. The doses were given using a syringe with attached gavage cannula. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulation was stirred continuously during dosing. The formulation for the high dose group was inverted in between the dosing of each cage in order to maintain homogeneity.

VEHICLE
- Justification for use and choice of vehicle: The test item is a black powder and is not soluble in organic solvents or in water (aqueous solubility measured at 54 µg/L).
- Concentration in vehicle: 10, 30 and 100 mg/mL for dose level 100, 300 and 1000 mg/kg bw/day, respectively
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Due to the poor solubility in solvent and water, and the difficult nature of the test item, it is not amenable to standard chromatography methods, and there are no alternative methods of analysis.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

gestation day 6 to 20

Frequency of treatment:

daily

Duration of test:

15 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Previous studies indicated that the test item demonstrated minimal toxicity to rats. In an acute oral toxicity study, the LD50 was >2000 mg/kg bw with no treatment-related signs of toxicity. The test item is also cationic and insoluble in water, which suggested there would be low absorption through the oral exposure route. The collective information on toxicity and the physical-chemical characteristics of the test item resulted in the decision to select 1000 mg/kg bw/day to be the highest dose level.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once at the start and once toward the end of the working day throughout the study
- Cage side observations checked: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during pretreatment (gestation day (GD) 4) and daily during the dosing period from GD 6.

BODY WEIGHT: Yes
- Time schedule for examinations: once during pretreatment (GD 4) and daily throughout the dosing period from GD 6

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: ovary, uterus

OTHER: Thyroid Hormone Analysis was performed ((T3) Triodothyronine, (T4) Thyroxine, (TSH) Thyroid Stimulating Hormone). Blood samples were taken from all animals on GD 20.

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: placenta (size, colour or shape) - only abnormalities were recorded

Fetal examinations:

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

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and have been reported at the 1%, and 5% levels. Descriptive statistics number, mean and standard deviation have been reported whenever possible. Inferential statistics were performed when possible, but excluded any group with less than 3 observations.
Parametric/Non-Parametric: Levene’s test was used to assess the homogeneity of group variances. Datasets with at least 3 groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was. If the overall F-test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Indices:

Pregnancy Rate = No. of animals pregnant/No. of animals in cohabitation x 100

Pre-Implantation Loss = (No. of corpora lutea – no. of implants)/No. of corpora lutea x 100

Post-Implantation Loss = (No. of implants – no. of live fetuses)/No. of implants x 100

Sex Ratio (% males) = No. male fetuses/Total no. of fetuses x 100

Litter % of Fetuses with Abnormalities = No. of fetuses in litter with a given finding/No. of fetuses in litter examined x 100

Historical control data:

none provided

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Black faeces were observed at a dose level of 300 or 1000 mg/kg bw/day. This was considered due to the colour of the test item in the dose formulation and considered a non-adverse effect. In addition, ploughing of the cage shavings was observed in all dose groups including the controls. As ploughing is indicative of a dislike of taste, is it considered that this finding was likely due to the rats disliking the taste of corn oil and was considered not to be associated with test item administration.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dose-related statistically higher body weight gains were observed from Day 18 to 21 at dose levels of 300 or 1000 mg/kg bw/day (28% and 33%, respectively) when compared with the control group (see table 1 in "any other information on results incl. tables" for details). These findings correlated with the increased food consumption. However, findings did not have a biological relevance and are therefore considered not to be adverse.
There were no other effects on body weights or body weight gains.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 300 or 1000 mg/kg bw/day, higher food consumption was observed during GD 17 to 21 in comparison to the control group. From GD Day 18-19 a statistical increase of 18% or 17%, respectively and on GD Day 20-21 a statistical increase of 52% or 60% was observed. These findings did not have a biological relevance and therefore considered not to be adverse (see table 2 in "any other information on results incl. tables" for details).
There were no other effects on food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

At dose levels up to and including 1000 mg/kg bw/day, there were no test substance-related effects on gravid uterine weights or corrected body weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At dose levels up to and including 1000 mg/kg bw/day, there were no test substance-related gross pathology findings in the pregnant rats.
Single findings such as abnormal appearance of the placenta or dark focus on thymus were considered to be incidental findings which can occur in this species and were therefore considered to be unrelated to test substance administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in the thyroid hormone parameters when compared with the controls.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day, the total number of pre and post-implantation loss was higher when compared with the controls by 455% and 227%, respectively. The elevated implantation means were considered to be a result of high losses in individual animals (3505, 3513 or 3517) that were also noted to have an initial lower corpora luteum count. The remaining animals in Group 3 were comparable to the controls and or within background findings, as were the mean live and early resorption incidences. Furthermore, there were no dose-related pattern of the pre and post-implantation losses. Therefore, these findings were considered to be sporadic and unrelated to test item administration.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

A total number of dams with resorptions was noted to be higher at all dose groups when compared with control dams. However, these findings were noted to be within the expected ranges of variations in the background findings, whereas the control values were noted to be lower than the expected ranges of variations of the background findings. The number of resorptions in the females dosed at 100, 300 or 1000 mg/kg bw/day (early on) were minimal and had no impact on the total number of live fetuses. Therefore, these observations were considered incidental and unrelated to Organolignite administration.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

not examined

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed up to the highest dose tested

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed up to the highest dose tested

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Summary of Body Weight Gains (g)

Sex: Female		Day(s) Relative to Mating (Litter: A)
		6 → 9 [G]	6 → 12 [G1]	6 → 15 [G1]	6 → 18 [G1]	18 → 21 [G]	6 → 21 [G]
0	Mean	15.7	34.0	53.6	89.8	30.6	120.4
mg/kg/day	SD	6.1	9.2	11.9	12.1	11.4	18.8
	N	24	24	24	24	24	24
Group 1		-	-	-	-	-	-
100	Mean	15.0	34.4	53.5	88.3	35.4	123.7
mg/kg/day	SD	5.9	7.7	10.0	13.5	7.0	18.8
	N	24	24	24	24	24	24
Group 2		-	-	-	-	-	-
300	Mean	16.1	34.0	54.1	90.1	39.1*	129.3
mg/kg/day	SD	4.0	7.1	9.2	14.9	8.4	21.9
	N	24	24	24	24	24	24
Group 3		-	-	-	-	-	-
1000	Mean	15.1	36.6	56.1	92.7	40.6*	133.3*
mg/kg/day	SD	7.4	6.2	6.7	9.3	7.0	10.0
	N	23	23	23	23	23	23
Group 4		-	-	-	-	-	-

[G] - Kruskal-Wallis & Dunn: * : p <= 0.05

[G1] - Anova &Dunnett

Table 2: Summary of Food Consumption: Daily Food Consumption Per Animal (g)

Sex: Female		Day(s) Relative to Mating (Litter: A)
		19 → 20 [G]	20 → 21 [G1]
0	Mean	15.5	10.8
mg/kg/day	SD	2.8	4.8
	N	12	12
Group 1		-	-
100	Mean	16.8	12.7
mg/kg/day	SD	3.0	2.5
	N	12	12
Group 2	%Diff	8.3	18.2
300	Mean	18.6*	16.3*
mg/kg/day	SD	2.1	2.8
	N	12	12
Group 3	%Diff	19.9	51.9
1000	Mean	19.0**	17.2**
mg/kg/day	SD	2.5	2.5
	N	11	11
Group 4	%Diff	22.3	59.8

[G1] - Kruskal-Wallis & Dunn: * : p <= 0.05; **: p <= 0.01

[G] - Anova &Dunnett: * : p <= 0.05; **: p <= 0.01

Table 3: Summary of Maternal Performance and Mortality

Sex: Female Day(s) Relative to Mating (Litter: A)		0mg/kg bw /day Group 1	100 mg/kg bw/day Group 2	300 mg/kg bw/day Group 3	1000 mg/kg bw/day Group 4
Group Size - Females		24	24	24	24
Number of Females Pregnant [f]	N+ve	24	24	24	23
	%	100.0	100.0	100.0	95.8
Female with Live Fetuses [f]	N+ve	24	24	24	23
	%	100.0	100.0	100.0	100.0
Female with all Nonviable [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Placenta exam Normal [f]	N+ve	24	23	23	22
	%	100.0	95.8	95.8	95.7
Fem. Euthanized Preterminally [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Female with Resorptions [f]	N+ve	3	6	5	7
	%	12.5	25.0	20.8	30.4
f : Fisher‘ Exact test

Table 4: Summary of Overian and Uterine Examination and Litter Examination and Litter Observation

Sex: Female   Day(s) Relative to Mating (Litter: A)		0mg/kg bw/day   Group 1	100 mg/kg bw/day   Group 2	300 mg/kg bw/day   Group 3	1000 mg/kg bw/day   Group 4
Female with Live Fetuses [f]	N+ve	24	23	24	22
	%	100.0	100.0	100.0	100.0
Number of Corpora Lutea [k]	Mean	13.4	13.9	13.9	13.6
	SD	1.1	2.6	2.7	1.5
	N	24	23	24	22
	%Diff	-	3.7	4.0	1.6
Number of Implantations [k]	Mean	13.1	13.2	12.3	12.9
	SD	1.2	2.5	3.3	1.7
	N	24	23	24	22
	%Diff	-	1.0	-5.7	-1.3
Pre-implantation Loss (%) [k1]	Mean	2.19	4.48	12.16	5.07
	SD	4.09	5.81	17.97	7.05
	N	24	23	24	22
	%Diff	-	104.58	455.27	131.40
Total Number of Resorptions [k]	Mean	0.2	0.3	0.4	0.4
	SD	0.5	0.6	1.0	0.6
	N	24	23	24	22
	%Diff	-	108.7	150.0	118.2
Number of Early Resorptions [k]	Mean	0.2	0.3	0.4	0.4
	SD	0.5	0.6	1.0	0.6
	N	24	23	24	22
	%Diff	-	108.7	150.0	118.2
Number of Late Resorptions [I]	Mean	0.0n	0.0n	0.0n	0.0n
	SD	0.0	0.0	0.0	0.0
	N	24	23	24	22
	%Diff	-	-	-	-
Total Number of Fetuses [k]	Mean	12.9	12.9	11.9	12.5
	SD	1.4	2.4	3.6	1.7
	N	24	23	24	22
	%Diff	-	-0.4	-7.7	-2.9
Number of Live Fetuses [k]	Mean	12.9	12.9	11.9	12.5
	SD	1.4	2.4	3.6	1.7
	N	24	23	24	22
	%Diff	-	-0.4	-7.7	-2.9
Number of Live Male Fetuses [k]	Mean	6.7	6.6	6.0	6.6
	SD	1.6	1.6	2.4	1.7
	N	24	23	24	22
	%Diff	-	-1.5	-10.0	-0.5
Number of Dead Fetuses [I]	Mean	0.0n	0.0n	0.0n	0.0n
	SD	0.0	0.0	0.0	0.0
	N	24	23	24	22
	%Diff	-	-	-	-
Post-implantation Loss (%) [k]	Mean	1.31	2.51	4.28	2.79
	SD	3.75	4.79	12.13	4.47
	N	24	23	24	22
	%Diff	-	91.86	226.79	113.40
Live Male Fetus/Litter (%) [k]	Mean	52.21	51.59	52.11	52.96
	SD	13.61	11.46	15.38	11.62
	N	24	23	24	22
	%Diff	-	-1.18	-0.19	1.42
Number of Live Female Fetuses [k]	Mean	6.3	6.3	5.9	5.9
	SD	2.2	2.1	2.7	1.7
	N	24	23	24	22
	%Diff	-	0.9	-5.3	-5.5
Mean Fetal Weight (both) (g) [G]	Mean	5.42	5.60	5.68 *	5.66
	SD	0.32	0.39	0.35	0.31
	N	24	23	24	22
	%Diff	-	3.32	4.80	4.35
Mean Fetal Weight (M) (g) [G]	Mean	5.55	5.73	5.81	5.82
	SD	0.37	0.42	0.42	0.36
	N	24	23	24	22
	%Diff	-	3.32	4.69	5.01
Mean Fetal Weight (F) (g) [G]	Mean	5.30	5.48	5.56 *	5.47
	SD	0.32	0.35	0.32	0.29
	N	24	23	24	22
	%Diff	-	3.27	4.91	3.22
Live Sum Fetal Weight (g)	Mean	69.875	71.499	67.615	70.601
	SD	6.759	10.772	20.356	7.387
	N	24	23	24	22
[f] - Fisher's Exact [k], [k1] - Kruskal-Wallis & Dunn [I] - n - Inappropriate for statistics [G] - Anova & Dunnett: * = p ≤ 0.05

Applicant's summary and conclusion

Conclusions:

In conclusion, administration of Organolignite by once daily oral gavage to pregnant Sprague Dawley rats from Gestation Day 6 to 20 at dose levels up to 1000 mg/kg bw/day had no adverse effects on any of the endpoints evaluated. In the pregnant females, there were no adverse effects on clinical observations, body weights, body weight gains or food consumption and no effects on thyroid hormone analysis, gross necropsy findings or any maternal parameter. Fetal examination did not reveal any adverse effects of Organolignite on any of the fetal endpoints. Based on these results, the maternal and fetal no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.

Executive summary:

The objective of this study was to determine the potential toxicity of the chemical Organolignite (an emulsifier in drilling mud) on prenatal development in the rat, following oral gavage dosing from Gestation Days 6 to 20. 24 female Sprague-Dawley rats received vehicle only (corn oil), 100, 300 or 1000 mg/kg bw/day test substance via oral gavage.

The following parameters and endpoints were evaluated in this study: clinical observations, body weights, food consumption, clinical pathology parameters (thyroid hormone analysis), gross necropsy findings, gravid uterine weights and corrected body weights, examination of pregnancies on Day 21 of gestation, ovarian and uterine findings, and fetal examinations (external abnormalities, visceral examination, sex and skeletal examinations, and body weights).
Black faeces were noted at dose levels of 300 or 1000 mg/kg bw/day. This finding was considered due to the colour of the test item in the dose formulation and was a non-adverse finding. Ploughing was observed in all dose groups including the controls. This was considered to be related to palatability as the formulation was corn oil. A significantly higher body weight gain was observed on Gestation Day 18-21 at 300 or 1000 mg/kg/day (by 28% or 33%, respectively). A correlating higher food consumption was also noted these days (up to +60%). However, these findings did not have a biological relevance and were considered non-adverse. There were no other effects on clinical observations, body weights, body weights gains or food consumption and no effects on thyroid hormone analysis, gross necropsy findings, examination of pregnancies on Day 21 of gestation, ovarian and uterine findings or gravid uterine weights. There were no adverse fetal effects resulting from test substance administration to dams. Fetal sex, body weights and the type of distribution of all fetal malformations, abnormalities and variations, including those indicating the extent of skeletal ossification, were similar in all groups and/or due to individual variation.
In conclusion, administration of Organolignite by once daily oral gavage to pregnant Sprague Dawley rats from Gestation Day 6 to 20 at dose levels up to 1000 mg/kg bw/day had no adverse effects on any of the endpoints evaluated. In the pregnant females, there were no adverse effects on clinical observations, body weights, body weight gains or food consumption and no effects on thyroid hormone analysis, gross necropsy findings or any maternal parameter. Fetal examination did not reveal any adverse effects of the test substance on any of the fetal endpoints. Based on these results, the maternal and fetal no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg bw/day.