Tall oil, compd. with ethanolamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 August 2012 to 12 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, compd. with ethanolamine

EC Number:

268-069-1

EC Name:

Tall oil, compd. with ethanolamine

Cas Number:

68002-54-0

Molecular formula:

Not applicable. It is not considered possible to assign a specific molecular formula to a UVCB substance.

IUPAC Name:

Tall oil, compd. with ethanolamine

Test material form:

liquid: viscous

Details on test material:

- Appearance: Amber/dark orange viscous liquid/fluid gel
- Storage conditions: Room temperature, protected from light

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han)
- Age at study initiation: On the first day of dosing, animals were 71 to 78 days of age.
- Weight at study initiation: On the first day of dosing, the males weighed 283 g to 333 g and the females weighed 184 g to 219 g.
- Housing: The animals were housed in groups of 5 by sex until pairing and for males post-pairing. For pairing, one male and one female were housed together and mated females were housed individually during gestation and with their litter during the lactation period. Males were housed in grid-floor cages suspended over paper-lined trays. During the pre-pairing and mating periods females were housed in grid-floor cages suspended over paper-lined trays and following mating females and their litter were housed in solid-floor cages with appropriate bedding provided.
- Diet (e.g. ad libitum): A pelleted rodent diet was supplied ad libitum.
- Water (e.g. ad libitum): Mains tap water (in bottles) was freely available.
- Acclimation period: The animals were acclimatised within the study room for 14 days before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C (target range 21 ± 2 °C).
- Humidity (%): 46 to 79 % (target range 55 ± 15 %).
- Air changes (per hr): Not reported - the room was air-conditioned.
- Photoperiod (hrs dark / hrs light): The study room was illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dark.

IN-LIFE DATES: From: 2 August 2012 To: 12 November 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Elga UHP water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The required amount of test material was weighed and approximately 80 % of the vehicle was added. The mixture was stirred and heated to approximately 50 ± 5 °C, as required, until a visibly homogenous liquid was formed. The formulation was allowed to cool to ambient temperature, followed by further addition with the vehicle to the exact volume. Due to the lack of any stability information, the formulations were prepared daily and used within 3 hours of preparation.

VEHICLE
- Dose volume: 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

FORMULATION SAMPLING
Samples were taken from each formulation. The formulations (including controls) prepared for use on the first day of dosing and subsequent formulations prepared for use during Week 6 were analysed to assess achieved concentrations. All remaining samples were stored frozen (approximately -18 °C).

ANALYSES OF TEST MATERIAL FORMULATIONS
Samples from each designated formulation, including the vehicle used to dose the control group, were analysed for the test material. During Week 6, individual achieved concentrations from the formulations prepared for use on Day 40 of the study (Groups 2 to 4) ranged from 113 to 126 % of the nominal. Since the majority of individual concentrations were above the acceptance criterion and contingency samples could not be analysed due to a lack of the frozen stability data, analysis of test material formulations prepared for use on Day 41 of the study (Groups 2 to 4 only) was performed.

METHOD OF ANALYSIS
The analytical procedure for the determination of the test material in water was conducted using UV/visible spectrophotometry using the test material, water and HPLC grade methanol.
- Instrument: Unicam UV/visible spectrophotometer

PROCEDURE
Linearity of Detection
The linearity of detection was assessed from triplicate measurements of six standards prepared at concentrations over the calibration range of the assay. The standard concentration and response data were subjected to linear regression analysis and the assay considered linear if the correlation coefficient (r) was no less than 0.99.
The linear correlation coefficient over the calibration range 20.32 to 81.80 µg/mL of the test material was 0.9968.

Precision of Detection
The precision of detection was assessed over the linear range from the coefficient of variation of the response factors (response/concentration) of all standards analysed in triplicate.
The precision of detection at the mid-point of the linear range was assessed from the coefficient of variation of the responses of a mid-point standard analysed six times consecutively. The precision of detection was considered acceptable if the coefficients of variation were no greater than 7.5 % for each assessment.
The precision of detection over the linear range was 3.4 %. The precision of detection at the mid-point of the linear range was 0 %.

System Stability
The stability of the system was assessed from the coefficient of variation of the responses of a mid-point standard, analysed six times interspersed throughout the run, and was considered acceptable if no greater than 7.5 %.
The stability of the system was 0.1 %.

Specificity
The specificity of the assay was assessed from the analysis of control samples and reagent blanks and was considered acceptable if the absorbance was no greater than 5 % of that of the lowest concentration standard.
No response that was attributed to the test material was observed in the control samples or reagent blanks.

Intra-Assay Accuracy and Precision
The intra-assay accuracy at each concentration, expressed as the mean accuracy values (n=3) was assessed from the analysis of triplicate samples of the test material in water, at concentrations of 10 mg/mL and 200 mg/mL. The assay was considered accurate if the individual and the mean measured concentrations of the test material were between 85 and 115 % of their nominal values. The intra-assay precision, expressed as the coefficient of variation of the individual accuracy values at each concentration, was considered acceptable if no greater than 10 %.
The individual accuracy values at 10 mg/mL were between 85 and 89 % with a mean value of 87 % and a precision value of 2.5 %. The individual accuracy values at 200 mg/mL were between 85 and 87 % with a mean value of 86 % and a precision value of 1.1 %.

Inter-Assay Accuracy and Precision
Assay accuracy and precision, were repeated on a separate occasion, using freshly prepared solutions and reagents, to establish the effects of such changes.
The inter-assay accuracy at each concentration, expressed as the mean accuracy values (n=6) obtained over two occasions, was considered acceptable if the individual and the mean measured concentrations of the test material were between 85 and 115 % of their nominal values.
The inter-assay precision at each concentration, expressed as the coefficient of variation of the individual accuracy values (n=6) obtained over two occasions, was considered acceptable if no greater than 15 %.
The individual accuracy values at 10 mg/mL were between 85 and 95 % with a mean value of 90 % and a precision value of 4.3 %. The individual accuracy values at 200 mg/mL were between 85 and 89 % with a mean value of 88 % and a precision value of 2.0 %.

RESULTS OF FORMULATION ANALYSIS
Formulations used to dose animals during Week 1 of the study were considered accurate since the measured concentrations were within 15 % of their nominal values which fulfilled the acceptance criterion.
The majority of samples from test formulations used to dose animals in Groups 2, 3 and 4 on Day 40 of the study (Week 6) showed higher levels of the test material than the target values (by up to 26 %), which was outside the specified acceptance criterion. Test material formulations used to dose animals in Groups 2, 3 and 4 on Day 41 of dosing were also higher than their nominal values (by up to 30 %), which was again outside the specified acceptance criterion. Although no reason could be established for these higher values, considering these animals were dosed at higher dose levels than their target, this was considered to have no impact on the validity of the No Observed Adverse Effect Levels (NOAELs) determined in this study.
No test material was detected in vehicle used to dose animals in Group 1.

Details on mating procedure:

- M/F ratio per cage: After the pre-pairing dosing period, each female was paired with a treated male from the same group.
- Length of cohabitation: 14 days.
- Proof of pregnancy: During the pairing period, vaginal smears were taken daily, by lavage, until mating was confirmed either by sperm being found in the smear or by the number and nature of copulation plugs. The smear was examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present. The number of copulation plugs was also recorded to give an assessment of the mating activity of the animals. The day on which sperm were detected was referred to as Day 0 of gestation with the next day classified as Day 1 of gestation.
- After successful mating each pregnant female was caged (how): Mated females were housed individually during gestation and with their litter during the lactation period.

Duration of treatment / exposure:

The males were dosed for 14 days prior to, and during pairing, and until the day prior to necropsy. The females were dosed once daily for 14 days prior to, and during pairing, gestation and until Day 3 of lactation, inclusive.
Any female in mid-parturition at the time of dosing was not dosed on that day.

Frequency of treatment:

Once daily

Duration of test:

Females were sacrificed on Days 41 to 45; males were sacrificed on Day 55.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of results from a preliminary study. A high dose level of 1000 mg/kg/day was chosen, as this is the maximum dose level required by the guidelines. At this dose level in the preliminary study, clinical signs remained confined to excessive pre and/or post-dose salivation.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity. From the start of treatment, all animals were examined daily for clinical signs of toxicity or changes in behaviour and appearance.
During the treatment period, each main study animal was routinely checked pre-dose and soon after completion of dosing. On week days, additional observations were made approximately 1 hour after dosing and either approximately 4 hours after dosing or at the end of the working day (whichever was sooner). At weekends and public holidays, additional observations were made approximately 1 hour after dosing or at the end of the working day (whichever was sooner).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed clinical examination once each week, from the start of treatment. Standard arena observations (functional observations) were made cage side and outside the home cage, in a standard arena, for all animals. Observations were recorded once prior to the start of dosing and once weekly thereafter during the dosing period, at approximately the same time of day on each occasion (afternoon). On Day 40 of the study, these observations were not recorded for any females in mid-parturition or on Day 0 of lactation; they were instead recorded on Day 42 of the study for those females.

BODY WEIGHT: Yes
- Time schedule for examinations: Female body weights were recorded at the start of treatment and then at weekly intervals until the day of mating. Body weights for females were also recorded on Days 0, 7, 14 and 20 of gestation and then on Days 0 (where required for dose volume calculation), 1 and 4 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule: The amount of food consumed by the animals in each cage was recorded at weekly intervals during the pre-pairing dosing period. Food consumption of the females was also recorded over Days 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation and over Days 1 to 4 of lactation.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

URINALYSIS: No

OTHER: Neurobehavioural examinations, haematology and coagulation parameters and clinical chemistry parameters were evaluated.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes. The number of implantation scars and the number of corpora lutea for each female was recorded.

Fetal examinations:

F1 GENERATION
- Litter size and sex: The total litter size was recorded after completion of parturition and daily thereafter. Numbers of each sex were recorded on the same day as body weights were collected. The percentage of male pups, out of the total number of pups, was calculated for each litter. Day 0 of lactation was the day of completion of littering. The following day was considered to be Day 1 of lactation.
- Clinical observations: All pups were examined daily for clinical signs of toxicity or changes in behaviour and appearance.
- Mortality: Animals were examined twice daily for mortality and morbidity.
- Bodyweights: Pups were weighed individually on Days 1 and 4 of age.

Statistics:

Non-parametric methods were employed if it was considered that the assumptions required for parametric analysis did not hold.
No analyses were performed for variables for which there were less than five independent observations in the control group; when treated groups had fewer than five observations analyses were performed at the discretion of the statistician.
Data from each sex was analysed separately. Statistical significance was declared at the p<0.05 level in all cases and noted at the p<0.01 and p<0.001 levels.

COMPARISONS
Group 1 (control) against Groups 2, 3 and 4 (100, 300 and 1000 mg/kg, respectively)

STATISTICAL TESTS AND PARAMETERS
Data were processed to give group mean values and standard deviations where appropriate. Where the data allowed, the following methods were used for statistical analysis:

ANALYSIS OF VARIANCE AND WILLIAMS' TEST
(Non-parametric alternative: Kruskal-Wallis ANOVA and Shirley's Test)
Bodyweights and bodyweight gains, food consumption, number of implantation scars and number of corpora lutea, number of pups born, pup body weights and body weight gains (by sex and on litter basis), percentage of male pups, time course of mating, number of copulation plugs, duration of gestation, organ weights (absolute), relative organ weights (organ weights as a percentage of terminal body weight), clinical pathology data, grip strength and motor activity (timepoints 1 and 1 - 6 (total)).

ANALYSIS OF COVARIANCE
Motor activity (timepoints 2 to 6)

ANALYSIS OF VARIANCE
(Non-parametric alternative: Kruskal-Wallis Test)
Pre-dose body weights (Day 1)

COCHRAN ARMITAGE TREND TEST
Copulation and fertility indices

JONCKHEERE TERPSTRA TEST
Litter survival indices

Indices:

REPRODUCTIVE INDICES
Copulation index (%) = (number of animals mated / number of animals paired) x 100
Female fertility index (%) = (number of pregnant animals / number of animals mated) x 100
Male fertility index (%) = (number of males siring a pregnancy / number of paired males )x 100
Gestation index (%) = (number of pregnant females with live pups born / number of pregnant females) x 100

OFFSPRING VIABILITY INDICES
Live birth index (%) = (number of pups born alive / total number of pups born) x 100
Viability index 1 (%) = (number of pups alive Day 4 of age / number of pups born alive) x 100
Cumulative survival index (%) = (number of pups alive Day 4 of age / total number of pups born) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There was no mortality and there were no clinical signs considered to be related to the toxicity of the test material.

BODY WEIGHT AND FOOD CONSUMPTION
During the first week of the pre-pairing period, females given 1000 mg/kg/day showed a stasis in group mean bodyweight with half of these animals losing body weight; in comparison control animals gained weight during this period and the difference was statistically significant (p<0.001). Thereafter, an improvement was evident such that during the second week of dosing, the mean bodyweight gain for these females was higher than controls, albeit without achieving statistical significance. Group mean bodyweight gains for these females during the gestation and lactation periods remained unaffected. For females receiving 100 or 300 mg/kg/day of the test material, there was no effect of treatment on body weight gain during any stage of the study.

Females given 1000 mg/kg/day showed slightly lower food intake during the pre-pairing period and over Days 0 to 4 of gestation (p<0.05) when compared with controls. Throughout the remainder of the gestation period, the mean food intake values for this group of females were marginally lower than controls, albeit without achieving statistical significance. During lactation, food intake values for these females were similar to controls.
For animals given 100 or 300 mg/kg/day of the test material, food intake values were generally similar to controls throughout the study.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
There was no effect of treatment with the test material on the mean number of days taken to mate. With an exception of one female from the control group and one female from the group receiving 300 mg/kg/day, all other females mated within 4 days of pairing; the two exceptions were in persistent dioestrous prior to mating. The mean number of copulation plugs remained unaffected by treatment with the test material.

REPRODUCTIVE PERFORMANCE
There was no effect of treatment on fertility or mating performance.
There was no effect of treatment on the mean duration of gestation.
At 1000 mg/kg/day, group mean value for implantation scars was marginally but statistically significantly lower (p<0.05) than controls but this was considered coincidental; only 2 out of 10 animals had individual values slightly below controls. The mean number of corpora lutea for this group of females was also statistically significantly lower (p<0.05) than controls, however the recording of corpora lutea on Day 4 of lactation is considered inaccurate. Since post-partum oestrous occurs 24 to 48 hours after parturition in rats, during the lactation period there are two sets of corpora lutea; one from pregnancy and the other from the new oestrous. Since corpora lutea are counted under low magnification, it is impossible to distinguish between the two, and counting the corpora lutea may result in inaccurate numbers.

ORGAN WEIGHTS
At 1000 mg/kg/day, absolute and bodyweight-related liver weights were statistically significantly higher than controls (p<0.01). The majority of corresponding individual bodyweight-related liver weights for females, including controls, were above the background control data ranges. In the absence of any histopathology findings for the liver, the increases in liver weight were considered to be adaptive in nature and not indicative of toxicity.
At 300 or 1000 mg/kg/day, females also showed statistically significantly lower body weight-related heart weights than controls (p<0.05) but without any dose-relationship. The majority of individual values were within the background control data ranges and as there were no corresponding histopathology findings, this finding was considered not to be related to treatment with the test material.

GROSS PATHOLOGY
There were no macroscopic findings at necropsy considered to be related to the treatment with the test material.

HISTOPATHOLOGY
There was an equivocal change (erythrocytosis/erythrophagia) in the mesenteric lymph nodes for animals receiving 1000 mg/kg/day. No effect was noted for these parameters in the low or intermediate dose groups. In addition, an assessment of the bone marrow smears from control animals and those given 1000 mg/kg/day indicated that there was no effect of treatment on the assessed parameters.
A variety of spontaneous changes was noted in control and treated animals with no indication of an effect of treatment. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.

OTHER FINDINGS
HAEMATOLOGY AND COAGULATION
Leucocyte and lymphocyte values in all female groups given the test material were slightly lower than the respective control group, albeit without achieving statistical significance. In the absence of any associated histopathology findings, this was considered not to be of toxicological significance.
At 1000 mg/kg/day, females showed a marginal but statistically significant lowering in haemoglobin concentration (p<0.05) and slight prolongation of prothrombin time (p<0.001) when compared with controls. Individual haemoglobin concentrations were within the background control data range but respective prothrombin times were above these ranges. In the absence of any associated histopathology changes these findings were considered not to be toxicologically significant.

BLOOD CHEMISTRY
Statistically significantly higher mean plasma concentration of bile acids (p<0.05) was observed in females given 1000 mg/kg/day with respect to controls. At 100 or 300 mg/kg/day, a small number also showed higher concentrations of bile acids than controls. High individual variation seen in these animals may be due to the fact that these animals were not fasted prior to bleeding and the intergroup differences were considered unrelated to treatment.

FUNCTIONAL OBSERVATIONS
There were no clear effects of treatment on functional observations monitored throughout the study or sensory reactivity to visual, acoustic, tactile or proprioceptive stimuli monitored towards the end of the study.
- Grip Strength: Any differences between control and treated animals were non dose-related and individual variation was high amongst animals. These differences were considered not related to treatment with the test material.
- Motor Activity: Any differences between control and treated animals were non dose-related and individual variation was high amongst animals. These differences were considered not related to treatment with the test material.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The mean number of pups born per litter, sex ratio and mean live birth, viability and cumulative survival indices in all treated groups were comparable with controls.
There were no unscheduled pup deaths on the study. Group mean pup body weight gains between Days 1 and 4 of age were unaffected by maternal treatment with the test material.
A variety of clinical signs noted across all groups, including controls, were considered unrelated to maternal treatment with the test item.
There were no necropsy findings for pups considered to be related to maternal treatment with the test material.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1 F1 Pup Bodyweights (g) - Group Mean Values

Males

Dose (mg/kg/day)	N	Bodyweight†(g)		Bodyweight Gain†(g) (Day 1 to 4)
		1 Day of Age	4 Days of Age
0	10	6.9 ± 0.5	10.5 ± 0.7	3.7 ± 0.3
100	10	7.2 ± 0.9	11.0 ± 1.3	3.7 ± 0.5
300	10	6.5 ± 0.6	9.8 ± 0.9	3.3 ± 0.4
1000	10	7.0 ± 1.0	10.6 ± 1.6	3.5 ± 0.7

Females

Dose (mg/kg/day)	N	Bodyweight†(g)		Bodyweight Gain†(g) (Day 1 to 4)
		1 Day of Age	4 Days of Age
0	10	6.5 ± 0.3	10.1 ± 0.6	3.6 ± 0.4
100	10	6.9 ± 0.8	10.6 ± 1.2	3.7 ± 0.5
300	10	6.2 ± 0.7	9.5 ± 1.1	3.3 ± 0.5
1000	10	6.7 ± 0.9	10.2 ± 1.3	3.6 ± 0.5

†= Statistically analysed

N = Number of litters in mean

 

Table 4 pup Necropsy - F1 Generation

Parameter	Dose (mg/kg/day)
	0	100	300	1000
N	10	10	10	10
Number of Pups
Born Found dead/killed prematurely Missing (presumed cannibalised) Schedule sacrificed	123 0 0 123	118 0 0 118	129 0 0 129	112 0 0 112
Findings: Dead/Moribund/Sacrificed Pups
No abnormalities No milk in stomach Right testis & epididymis abnormal red colour	123 0 0	117 1 0	129 0 0	109 2 1

N = Total number of litters

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, in the Wistar strain rat, the No Observed Adverse Effect Level (NOAEL) for male and female repeated-dose toxicology was considered to be 300 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) for developmental effects was considered to be 1000 mg/kg/day.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422 under GLP conditions.

Three groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (UHP water) following the same dosing regimen as the treated animals and were used as controls. The males were dosed for 14 days prior to pairing, during pairing and until the day prior to necropsy. The females were dosed for 14 days prior to pairing, during pairing and gestation, and up to and including Day 3 of lactation.

There were no unscheduled deaths on the study and administration of the test material was well tolerated; some parental effects were seen on bodyweights, lymphocyte counts and bone marrow adipocytes however the magnitude of change was small.

There was no effect of treatment on fertility or mating performance. There was no effect of treatment with the test material on the mean duration of gestation. The mean number of pups born per litter, sex ratio and mean live birth, viability and cumulative survival indices in all treated groups were comparable with controls. There were no unscheduled pup deaths on the study.

Group mean pup body weight gains between Days 1 and 4 of age were unaffected by maternal treatment with the test material and there were no necropsy findings for pups considered to be related to maternal treatment with the test material.

Under the conditions of this study, in the Wistar strain rat, the No Observed Adverse Effect Level (NOAEL) for male and female repeated-dose toxicology was considered to be 300 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) for developmental effects was considered to be 1000 mg/kg/day.