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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) inhalation toxicity study, organolignite was administered to 10 Sprague-Dawley rats/sex/concentration by nose only exposure at concentrations of 0, 5, 50, or 300 mg/m³ for 6 hours per day, 7 days/week. There were no treatment-related effects on mating, pregnancy performance, fertility, maternal care, or pup performance. The reproductive NOAEC is 300 mg/m³ (equivalent to 0.3 mg/L) based on the lack of effect. In a 90 -day inhalation study (OECD 413) no abnormlities were observed on reproductive organs of male and female rats.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited, Margate, Kent, UK
- Age at study initiation: ca 6-7 weeks
- Weight at study initiation: 201-227 g males; 163-195 g females
- Housing: Housed in polycarbonate cages with stainless steel grid tops and solid bottoms. Animals were initially housed 2/cage by sex. Males were individually housed a few days prior to mating. After mating, males were re-housed with original cage mates.
- Diet (e.g., ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded) provided ad libitum
- Water (e.g., ad libitum): Public water supply provided ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70%
- Air changes (per hr): 10 air changes per hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Route of administration:

inhalation: dust

Type of inhalation exposure (if applicable):

nose only

Vehicle:

air

Details on mating procedure:

- M/F ratio per cage: 1:1 basis
- Length of cohabitation: Female remained with male until mating had occurred
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males were treated daily 2 weeks prior to mating, throughout mating, and until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated daily 2 weeks prior to mating, throughout mating, and up to Day 19 of gestation (ca. 6 weeks for treatment).

Frequency of treatment:

6 hr per day, 7 days per week

Dose / conc.:

5 mg/m³ air (nominal)

Dose / conc.:

50 mg/m³ air (nominal)

Dose / conc.:

300 mg/m³ air (nominal)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on the results for a previously performed study.
- Rationale for animal assignment (if not random): Cages were allocated treatment group by the use of randomly sequenced numbers in such a way that each complete rack contained representatives from all treatment groups.

Positive control:

The study did not include a positive control.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: One week prior to treatment and weekly thereafter
- Cage side observations included: posture/condition (i.e., prostration, lethargy, writhing, circling, breathing abnormalities, gait abnormalities, tremor, fasciculation, convulsions, biting, vocalizations, and piloerection); ease of removal from the cage; body temperature; condition of the eyes (i.e., pupillary function, miosis, mydriasis, exophthalmos, encrustation, and lacrimation) and coat; presence of salivation, and overall ease of handling.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: One week prior to treatment and daily thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: One week prior to treatment for both sexes. Thereafter, males were weighed twice weekly at the start of dosing until the end of the study. Females were weighed twice weekly at the start of dosing and daily from mating to termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
- Time schedule for examinations: Not reported

Oestrous cyclicity (parental animals):

Data not reported

Sperm parameters (parental animals):

Data not reported

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioral abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Yes. Pups dying prior to scheduled necropsy were examined externally and checked for the presence of milk in the study. A macroscopic examination of the tissues and organs of the cranial, thoracic, and abdominal cavities in situ. Pups and their dissected tissues were preserved in 10% neutral buffered formalin.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving males were sacrificed after mating and treatment for at least 4 weeks.
- Maternal animals: All surviving females were sacrificed between Day 5 and 7 of lactation.

GROSS PATHOLOGY: Yes (see Table 1)

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes; tissues identified in table 1 (except bone marrow smears) were processed from 5 males and 5 females from the control and high-dose groups.

Postmortem examinations (offspring):

SACRIFICE
- The F1 surviving offspring were scarified between Day 5 and 7 of lactation.

GROSS NECROPSY
- Surviving pups were examined externally for visible abnormalities. Those found to be externally normal were discarded. Pups dying prior to scheduled necropsy were examined externally and checked for the presence of milk in the study. A macroscopic examination of the tissues and organs of the cranial, thoracic, and abdominal cavities in situ. Pups and their dissected tissues were preserved in 10% neutral buffered formalin.

Statistics:

The following endpoints were analyzed using the 'F Max' test: selected body weight and food consumption data; all hematology, coagulation, and clinical chemistry; and selected functional observational battery and motor activity data. Homogeneous variances were analyzed using a parametric ANOVA. Pairwise comparisons were made using Fisher's F protected LSD method via Student's t test only if the overall F test was significant. Heterogeneous variances were initially transformed using the log or square root. If variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise compassions were made using chi squared protection.

Organ weight data were analyzed using the methods noted above and through analysis of covariance (ANCOVA) using terminal body weight as the covariate. Organ weight were also analyzed as a percentage of terminal body weight.

Reproductive indices:

Fertility index (male) = (number siring a litter)/number paired

Fertility index (female) = (number pregnant)/number paired

Gestation index = (number bearing live pups)/number pregnant

Offspring viability indices:

Birth index = (total number of pups born [live and dead])/number of implantation scars

Live birth index = (number of pups born live on Day 0 of lactation)/(total number born [live and dead])

Viability index = (number of pups live on Day 4 of lactation)/(number live on Day 0)

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY: Increased incidence of staining around the head, nose, eyes, muzzle, and ears for all treated animals. Mid- and high-dose animals also had staining on their coats.

ORGAN WEIGHTS: At the high-dose, a statistically significant increase in both absolute and relative lung weight was observed in high-dose males and females. At the mid-dose, there was a statistically significant increase in absolute lung weight for both males and females; however, only females exhibited a statistically significant increase in relative lung weight. No effects on organ weights were observed at the low dose for both males and females.

GROSS PATHOLOGY: There was an increased incidence of dark discoloration of the lungs for all high-dose males and females and dark discoloration of the bronchial lymph node for high-dose males (10/10) and females (9/10). High-dose females also showed an increased incidence of pale foci on the lungs (5/10) and dark discoloration of the mediastinal lymph node (5/10). Mid-dose males (8/10) and females (8/10) showed an increased incidence of dark discoloration of the lungs.

HISTOPATHOLOGY: NON-NEOPLASTIC: At the high-dose, the following treatment-related findings were noted: bronchiolo-alveolar pigmented macrophages/hyperplasia; multifocal alveolar foamy macrophage accumulation; and a higher severity and incidence of mononuclear cell infiltration. In the bronchial lymph node there were pigmented macrophages and increased cellularity. In the mediastinal lymph node, there were pigmented macrophages. A microscopic examination was not conducted at 0.05 and 0.005 mg/L.

Dose descriptor:

NOAEC

Remarks:

reproductive

Effect level:

300 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: lack of effect

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

VIABILITY (OFFSPRING): A slight, nontreatment-related difference in the number of live pups per litter on Days 0, 1, and 4 of lactation were observed.

BODY WEIGHT (OFFSPRING): At the low- and high-dose groups, litter weight on Day 4 of lactation were slightly lower than controls. This was mainly due to one dam in each group that had only one pup remaining. When these animals were removed for the group calculations, group male litter weights were comparable to the control values.

Dose descriptor:

NOEC

Generation:

F1

Effect level:

> 300 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effect

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) inhalation toxicity study, organolignite was administered to 10 Sprague-Dawley rats/sex/concentration by nose only exposure at concentrations of 0, 5, 50, or 300 mg/m3 for 6 hours per day, 7 days/week. There were no treatment-related effects on mating, pregnancy performance, fertility, maternal care, or pup performance. The reproductive NOAEC is 300 mg/m3 based on the lack of effect.

Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) inhalation toxicity study, organolignite was administered to 10 Sprague-Dawley rats/sex/concentration by nose only exposure at concentrations of 0, 5, 50, or 300 mg/m³ for 6 hours per day, 7 days/week. Males were treated daily 2 weeks prior to mating, throughout mating, and until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated daily 2 weeks prior to mating, throughout mating, and up to Day 19 of gestation (ca. 6 weeks for treatment).

 

There were no treatment-related effects on mating, pregnancy performance, fertility, maternal care, or pup performance. The systemic NOAEC is 300 mg/m³ (equivalent to 0.3 mg/L; i.e., the high dose) based on a lack of adverse effect. The reproductive NOAEC is 300 mg/m³ (i.e., 0.3 mg/L) based on the lack of effect.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

300 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

42

Species:

rat

Quality of whole database:

The quality of the whole database is high and meets the tonnage driven data requirements of REACH.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) via inhalation exposure, organolignite was administered to 10 Sprague-Dawley rats/sex/concentration by nose only exposure at concentrations of 0, 5, 50, or 300 mg/m³ for 6 hours per day, 7 days/week. Males were treated daily 2 weeks prior to mating, throughout mating, and until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated daily 2 weeks prior to mating, throughout mating, and up to Day 19 of gestation (ca. 6 weeks for treatment).

 

There were no treatment-related effects on mating, pregnancy performance, fertility, maternal care, or pup performance. The systemic NOAEC is 300 mg/m³ (equivalent to 0.3 mg/L; i.e., the high dose) based on a lack of adverse effects. The reproductive NOAEC is 300 mg/m³ (i.e., 0.3 mg/L) based on the lack of effect.

While this reproductive/developmental toxicity screening test (OECD 422) was well-conducted and scientifically sound, DNELs for this endpoint were not derived because screening studies are subject to some inherent deficiencies, most notably that a lack of observed effects does not necessarily confirm the absence of such effects. Coupled with the fact that two-generation (OECD 416) testing is being proposed, it is considered prudent to defer calculating DNELs for reproductive endpoints until definitive results from the two-generation study are available. This conclusion will be updated, if necessary, once information is available from the proposed study.

Effects on developmental toxicity

Description of key information

In a developmental toxicity study (OECD 414) organolignite was administered to 23 pregnant Sprague-Dawley rats/sex/dose via gavage at doses of 0, 100, 300, and 1000 mg/kg bw/day from gestation day 6 to 20. There were no treatment-related effects on maternal animals or fetuses. The maternal and developmental NOAEL is 1000 mg/kg bw/day based on the lack of adverse effects. This is supported by a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) inhalation toxicity study, in which organolignite was administered to 10 Sprague-Dawley rats/sex/concentration by nose-only exposure at concentrations of 0, 5, 50, and 300 mg/m³ for 6 hours per day, 7 days/week. There were no treatment-related effects on mating, pregnancy performance, fertility, maternal care, or pup performance. The developmental NOAEC is 300 mg/m³ (equivalent to 0.3 mg/L) based on the lack of effect.

Link to relevant study records

Reference

Reference 1

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

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

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

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

Dose / conc.:

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

Dose / conc.:

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

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.

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

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.

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

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

Key result

Abnormalities:

no effects observed

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

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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

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.

Reference 2

Endpoint:

developmental toxicity

Remarks:

screening study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 422

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited, Margate, Kent, UK
- Age at study initiation: ca 6-7 weeks
- Weight at study initiation: 201-227 g males; 163-195 g females
- Housing: Housed in polycarbonate cages with stainless steel grid tops and solid bottoms. Animals were initially housed 2/cage by sex. Males were individually housed a few days prior to mating. After mating, males were re-housed with original cage mates.
- Diet (e.g., ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded) provided ad libitum
- Water (e.g., ad libitum): Public water supply provided ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70%
- Air changes (per hr): 10 air changes per hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Route of administration:

inhalation: dust

Type of inhalation exposure (if applicable):

nose only

Vehicle:

air

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- M/F ratio per cage: 1:1 basis
- Length of cohabitation: Female remained with male until mating had occurred
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Males were treated daily 2 weeks prior to mating, throughout mating, and until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated daily 2 weeks prior to mating, throughout mating, and up to Day 19 of gestation (ca. 6 weeks for treatment).

Frequency of treatment:

6 hr per day, 7 days per week

Duration of test:

54 days

Dose / conc.:

5 mg/m³ air (nominal)

Dose / conc.:

50 mg/m³ air (nominal)

Dose / conc.:

300 mg/m³ air (nominal)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on the results for a previously performed study.
- Rationale for animal assignment (if not random): Cages were allocated treatment group by the use of randomly sequenced numbers in such a way that each complete rack contained representatives from all treatment groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: One week prior to treatment and weekly thereafter
- Cage side observations included: posture/condition (i.e., prostration, lethargy, writhing, circling, breathing abnormalities, gait abnormalities, tremor, fasciculation, convulsions, biting, vocalizations, and piloerection); ease of removal from the cage; body temperature; condition of the eyes (i.e., pupillary function, miosis, mydriasis, exophthalmos, encrustation, and lacrimation) and coat; presence of salivation, and overall ease of handling.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: One week prior to treatment and daily thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: One week prior to treatment for both sexes. Thereafter, males were weighed twice weekly at the start of dosing until the end of the study. Females were weighed twice weekly at the start of dosing and daily from mating to termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
- Time schedule for examinations: Not reported

Ovaries and uterine content:

Ovaries were weighed and examined; however, uterine content was not evaluated.

Fetal examinations:

Dams were allowed to litter. The following parameters were examined in F1 offspring: number pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, and weight gain. Pups surviving until scheduled necropsy were examined externally for visible abnormalities. Those found to be externally normal were discarded. Pups dying prior to scheduled necropsy were examined externally and checked for the presence of milk in the study. A macroscopic examination of the tissues and organs of the cranial, thoracic, and abdominal cavities in situ. Pups and their dissected tissues were preserved in 10% neutral buffered formalin.

Statistics:

The following endpoints were analyzed using the 'F Max' test: selected body weight and food consumption data; all hematology, coagulation, and clinical chemistry; and selected functional observational battery and motor activity data. Homogeneous variances were analyzed using a parametric ANOVA. Pairwise comparisons were made using Fisher's F protected LSD method via Student's t test only if the overall F test was significant. Heterogeneous variances were initially transformed using the log or square root. If variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise compassions were made using chi squared protection.

Organ weight data were analyzed using the methods noted above and through analysis of covariance (ANCOVA) using terminal body weight as the covariate. Organ weight were also analyzed as a percentage of terminal body weight.

Indices:

The following reproductive indices were calculated:

Fertility index (male) = (number siring a litter)/number paired

Fertility index (female) = (number pregnant)/number paired

Gestation index = (number bearing live pups)/number pregnant

The following offspring indices were calculated:

Birth index = (total number of pups born [live and dead])/number of implantation scars

Live birth index = (number of pups born live on Day 0 of lactation)/(total number born [live and dead])

Viability index = (number of pups live on Day 4 of lactation)/(number live on Day 0)

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
At necropsy, gross pathology results noted dark, discoloration of the lungs in mid- and high-dose females. At the high and mid dose, females also exhibited a statistically significant increase in absolute and relative lung weight. The microscopic evaluation showed pigmented macrophages in the lung at the high dose. At the high dose, area of bronchiolo-alveolar hyperplasia characterized by hypertrophic alveolar Type II pneumocyte cells forming a single layer outlining alveolar walls also was observed. There was no evidence, however, of any associated tissue damage (i.e., necrosis, degeneration, or fibrosis). Additionally there was no evidence indicative of a pre-neoplastic lesion.

Dose descriptor:

NOAEC

Effect level:

300 mg/m³ air

Based on:

test mat.

Basis for effect level:

gross pathology

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEC

Effect level:

300 mg/m³ air

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: No adverse effect

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) inhalation toxicity study, organolignite was administered to 10 Sprague-Dawley rats/sex/concentration by nose only exposure at concentrations of 0, 5, 50, or 300 mg/m3 for 6 hours per day, 7 days/week. There were no treatment-related effects on mating, pregnancy performance, fertility, maternal care, or pup performance. The developmental NOAEC is 300 mg/m3 (equivalent to 0.3 mg/L) based on the lack of effect.

Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) inhalation toxicity study, organolignite was administered to 10 Sprague-Dawley rats/sex/concentration by nose only exposure at concentrations of 0, 5, 50, or 300 mg/m³ for 6 hours per day, 7 days/week. Males were treated daily 2 weeks prior to mating, throughout mating, and until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated daily 2 weeks prior to mating, throughout mating, and up to Day 19 of gestation (ca. 6 weeks for treatment).

 

There were no treatment-related effects on mating, pregnancy performance, fertility, maternal care, or pup performance.T he systemic NOAEC is 300 mg/m³ (equivalent to 0.3 mg/L; i.e., the high dose) based on a lack of adverse effect. The reproductive NOAEC is 300 mg/m³ (i.e., 0.3 mg/L) based on the lack of effect.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The quality of the whole database is high and meets the tonnage driven data requirements of REACH.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

300 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

42

Species:

rat

Quality of whole database:

The quality of the whole database is high and meets the tonnage driven data requirements of REACH.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Two studies are available regarding developmental toxicity. A developmental toxicity study according to OECD 414 in rats via oral route and reproduction/developmental screening test (OECD 422) in rats via inhalation. The results of these studies are presented below in detail.

 

OECD 422:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) via inhalation exposure, organolignite was administered to 10 Sprague-Dawley rats/sex/concentration by nose only exposure at concentrations of 0, 5, 50 and 300 mg/m³ for 6 h per day, 7 days/week. Males were treated daily 2 weeks prior to mating, throughout mating, and until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated daily 2 weeks prior to mating, throughout mating, and up to Day 19 of gestation (ca. 6 weeks for treatment).

There were no treatment-related effects on mating, pregnancy performance, fertility, maternal care, or pup performance. The systemic NOAEC is 300 mg/m3 (equivalent to 0.3 mg/L; i.e, the high dose) based on a lack of adverse effect. The developmental NOAEC is 300 mg/m3 (i.e., 0.3 mg/L) based on the lack of adverse effects.

 

OECD 414:

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. Twenty-four female Sprague-Dawley rats received vehicle only (corn oil), 100, 300 and 1000 mg/kg bw/day test substance via oral gavage.

The following parameters and endpoints were evaluated: clinical observations, body weight, food consumption, clinical pathology parameters (including 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 foetal 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), compared with control. A correlating higher food consumption was also noted these days (up to +60%). However, these findings did not have a biological relevance and were therefore considered to be non-adverse. There were no other effects on clinical observations, body weight, body weight gain or food consumption. No effects were noted on thyroid hormone analysis, and the results of the gross necropsy , examination of pregnancies on Day 21 of gestation, ovarian and uterine examinations and gravid uterine weight were comparable between the control and treatment groups . There were no adverse foetal effects resulting from test substance administration to the dams. Foetal sex, body weights and the type of distribution of all foetal 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. Foetal examination did not reveal any adverse effects of the test substance on any of the foetal endpoints. Based on these results, the maternal and foetal no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg bw/day.  

Justification for classification or non-classification

Data from a developmental toxicity study, a screening reproductive/developmental toxicity study (OECD 422) and a 90 -day inhalation study with organolignite did not reveal any effects on reproductive or developmental parameters. Therefore, organolignite is not classified as toxic for reproduction under CLP EU Regulation 1272/2008 (GHS aligned).