3,3'-[methylenebis(oxymethylene)]bisheptane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 3 May 2012 to 25 January 2013

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1995

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-[methylenebis(oxymethylene)]bisheptane

EC Number:

244-815-1

EC Name:

3,3'-[methylenebis(oxymethylene)]bisheptane

Cas Number:

22174-70-5

Molecular formula:

C17H36O2

IUPAC Name:

3,3'-[methylenebis(oxymethylene)]diheptane

Test material form:

liquid

Details on test material:

Batch No.: 1204051500
Purity: 99.766 % (w/w)
Impurities: 2-Ethylhexanol (0.0826 %); Formaldehyde (not found); Water (0.0370 %)
Storage: Store in supplied bottle in dark at the temperature under 25°C. Provide local exhaust or general room ventilation. Keep container closed when not in use. Keep away from heat.
Safety precautions: Do not breathe gas, fumes, vapour and spray. Use personal protective equipment (goggles, gloves).
Stability/Expiration: 3 years / 05.04.2015

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lambiotte & Cie - 1204051500
- Purity: 99.766%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in supplied bottle in dark at the temperature under 25°C. Provide local exhaust or general room ventilation. Keep container closed when not in use. Keep away from heat.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: test substance is homogenous and stable at least for 120 minutes in vehicle (Olivae oleum raffinatum) at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Details on species / strain selection:

the preferred rodent species according to the guidelines is the rat; the test facility has long experience with this species

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10 wks
- Fasting period before study: no
- Housing: Animals were housed in individually ventilated cages, 2 rats of the same sex in one polysulfone cage (floor area – 900 cm2) containing sterilised clean shavings of soft wood. During mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother.
- Diet: ad libitum, Complete pelleted diet for rats and mice in SPF breeding - ST 1 BERGMAN was used, manufacturer: Ing. Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using
- Water: ad libitum, Free access to drinking water (ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry. Water was sterilised before using.
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+3°C
- Humidity (%): 30-70%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 27/06/2012 To: 26/08/2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Remarks:

Pharmaceutical quality

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The concentrations of suspension at all dose levels was adjusted to ensure the administration of 1 mL per 100 g of body weight. The test substance was administered in olive oil (pharmaceutical quality). The application form (test substance in vehicle) was prepared daily just before administration and it was mixed by magnetic stirrer (500 rpm for 10 minutes).

VEHICLE:
- Lot/batch no. (if required): 5211201
- Purity: 12/2013

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
- Length of cohabitation: 2 weeks

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The treated groups were administered daily for the period: males and females – 2 weeks prior to the mating period and 2 weeks during the mating period. Pregnant females were administered during pregnancy and till 4th day of lactation. Males were administered till 28th day of the study, females showing no-evidence of copulation till 54thday of the study and non-pregnant females till the 25th day after confirmed mating. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

160 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for study – 160, 400 and 1000 mg/kg/day were chosen with respect to the results of the dose-range finding experiment

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily before and during application

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, after application at the same time every day (12.00 - 14 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia.
Weight increment was computed as an mean per group per day (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE:
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males mean values were calculated for each week of the study (except of mating period). Food consumption for animal/day was calculated from mean values of each group.
The same way of calculation of mean food consumption was used for females in premating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Non pregnant and aborted females (females without parturition) were not included in calculation of mean food of pregnant females.

Oestrous cyclicity (parental animals):

vaginal smears; daily in mating period

Sperm parameters (parental animals):

Parameters examined in all male parental: sperm motility ans sperm morphology, testis weight, epididymis weight, sperm motility, sperm morphology

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight, changes in behavioral abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Dead pups were sexed and externally examined; the stomach was examined for the presence of milk. Pups killed on the 4th day of lactation were sexed and subjected to external examination of the cranium and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.

Postmortem examinations (parental animals):

Parental males were killed at the end of the administration period – after 28 days of administration. Parental females were killed on the 4th day of lactation. Mated females without delivery were killed 26th day after confirmed mating.
Then they were macroscopically examined for any pathological changes with special attention to the organs of the reproductive systems. All macroscopic abnormalities were recorded.

Postmortem examinations (offspring):

external examination of the cranium and to macroscopic examination of the thoracic and abdominal tissues and organs

Statistics:

The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of body weight, biometry of organs and number of pups. Control group with vehicle was compared to three treated groups.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the summary tables.

Reproductive indices:

see table 1

Offspring viability indices:

see table 2

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
In treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. During the application period no clinical changes were observed in control males.
In animals of all dose levels the following changes were detected during the health condition control and clinical observation since the 2nd week of application: congestion of mucous membranes and skin of apical part of the body.

Females:
In treated females of all dose levels no signs of disease were recorded during the check-in and acclimatisation period. During the application period no clinical changes were observed in control females.
In animals of all dose levels during the health condition control and clinical observation since the 2nd week of application were detected: congestion of mucous membranes and skin of apical part of the body. In females of the dose level of 1000 mg/kg/day salivation and piloerection were detected. The salivation was detected since the 5th week of application in two females and piloerection was observed since the 6th week of application in one female

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the whole study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males: Statistical analysis was performed for necropsy body weights. The statistically significant differences were not detected. Body weights of control males and males of the lowest and the middle dose level were quite balanced during the application period.
The mean body weight of treated males at the dose levels 1000 mg/kg/day was lower in comparison with the control males during the application period. The weight increments in males of the highest dose level were markedly lower compared to control in the 1st and the 3rd week of the application period.
No statistically significant changes were found on probability level 0.05 (ANOVA test).
See table 3

Females:
Before mating period
The mean body weight and the mean body weight increments of the control females and treated females were well balanced (slightly increased in the 2nd week in treated females).
Pregnancy
Females without parturition (non pregnant or aborted females) were not included in the evaluation of mean body weight increments during pregnancy.
The mean body weight of treated mothers at all dose levels were similar in comparison with control whereas mean body weight increments of the females at the dose levels 1000 mg/kg/day were slightly decreased against control females on the 14th and 20th day of pregnancy.
Lactation
Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. The mean body weight increments of treated mothers at all dose levels were decreased against control but this difference was not dependent on dose level.
No statistically significant changes were found on probability level 0.05 (ANOVA test).
See table 4

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Males:
The mean food consumption of treated males at all dose levels was decreased compared to the food consumption of the control males.
See table 5

Females:
Statistical analysis was performed for mean food consumption in pregnancy (7th – 20th day) and in lactation.
Pre-mating period
The mean food consumption of females at the middle dose level was lower than in control females.
Pregnancy
Females without parturition (non pregnant, aborted, non paired females) were not included in evaluation of food consumption during pregnancy.
The mean food consumption of mothers at all dose levels was decreased against control mothers (statistically significant at the 2nd week of pregnancy).
Lactation
Only mothers (without non pregnant, aborted, non paired females) were included in evaluation of food consumption during lactation period.
The mean food consumption of treated females at all dose level was statistically significantly lower than in control females (without dose dependence).
See table 6

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Histopathological examination of the male reproductive system was performed at the control group and at the highest dose. The incidence of affected males is expressed in numeric form and ranged in sequence of the dose levels of 0 -1000 mg/kg/day further in the text.
Slight lymphocyte infiltration of the interstitium and epithelium in the epididymis was observed in 0-2 males. The germ cells in lumen of tubules in epididymis were detected in 2-2 males.
In the testes degeneration and/or atrophy of germinative epithelium in testicular tubules (less than 10 % tubules) were detected in 3-1 males. The exfoliation of germ cells to lumen of tubules were observed in 6-5 males (to 10 % tubules), in 0-1 male (more than 10 % tubules).
Lymphocyte infiltration of the interstitium was described in the prostate gland of 1-2 males. Atrophy of alveolar epithelium was detected in prostate gland of 0-1 male. The focal oedema of interstitium occurred in the prostate gland of 1-1 males. The focal desquamation of epithelium was observed in the seminal vesicles of 0-1 male, in the coagulating gland of 0-1 male. Presence of cysts in adenohypophysis was detected in pituitary gland of 2-0 males.

Histopathological examination of the liver and kidneys was performed at the middle and at the highest dose levels and is expressed in numeric form and ranged in sequence of the dose levels of 400-1000 mg/kg/day further in the text. The more often observed histopathological finding was hepatocellular hypertrophy (in 12-10 males). The focal infiltration and/or fibrosis were detected in liver of 2-3 males; vacuolation of hepatocytes in 1-0 male.
The tubular hypertrophy in cortex of kidneys was determined in 4-8 males, haemorrhage in kidneys in 0-1 male.
The reproductive organs were not examinated in males at the lowest and middle dose level groups. Microscopically examination of other organs was performed only in such dose level and for such organs where macroscopical findings organs were observed.
See table 11

Females:
The incidence of affected females is expressed in numeric form and ranged in sequence at the dose levels of 0-160-400-1000 mg/kg/day further in the text.
Histopathological examination of the organs of female genital tract (vagina, cervix, uterus, ovary) showed changes only in the uterus – in both control and treated mothers: signs of previous gravidity (focal accumulation of macrophagic elements - lipophages and siderophages in mesometrium) were recorded in 10-7-9-7 females; siderophages in mucosa in 7-1-8-5 females. Hydrometra (accumulation of ovulatory intraluminal fluid – spontaneous change during the oestrous cycle) were detected in 0-1-0-1 females. Acute endometritis (most propably of spontaneous origin) was observed in 0-0-0-1 female. Organizing hematoma was detected in 3-3-1-3 females.
Small myoepithelial adenoma (most propably of spontaneous origin) in mammary gland was found in 0-0-0-1 female.
The observation of the liver was performed at the middle and at the highest dose levels and is expressed in numeric form and ranged in sequence at the dose levels of 400-1000 mg/kg/day further in the text. In the liver parenchyma were found hypertrophy (in 6-10 females) accompanied by the presence of extramedullary hemapoiesis in 3-3 females.
See table 12

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

the investigation did not reveal serious damage of sperms quality in treated males versus control. The test substance did not influence sperm motility. The percentual proportion of morphologically changed sperms was slightly increased in all treated males. The most frequent changes were bent tails in sperms. In spite of sporadic change in sperm morphology the fertility of treated males was not affected. But the animals in the reproduction screening study are treated for the time period which is shorter than the duration of whole spermatogenic cycle so that an effect on spermatogenesis may not have had adequate time to become evident as reduced sperm counts that affect fertility.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

ability of males and females to successfully mate and achieve pregnancy was not adversely influenced by the test substance treatment. The number of females achieving pregnancy and duration of pregnancy were unchanged in treated groups compared to control. The duration of mating was slightly longer at the highest dose level.
The fertility index was decreased without dose dependence and the alteration was observed also in control group. In fertility index may be difficult to determinate the affected sex when both sexes were dosed furthermore males were dosed for less than the duration of the spermatogenic cycle so reproductive effect may not be manifested in the fertility index.
The mating index and gestation index were decreased at the highest dose level. The numbers of aborted females were increased with doses. At the highest dose level 2 aborted females, at the middle dose level 1 aborted female were observed.
The numbers of corpora lutea and numbers of implantations were decreased at the highest dose level (it cohered to lower number of pups at this dose level) but manifested only statistically insignificant differences. Slight decreased of pre-implantation losses, post-implantation losses and post-natal losses at the highest dose level associated with lower number of corpora lutea, implantations and number of live pups.
The survival index – an important endpoint reflects the ability of the pups to survive – was not changed.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: none

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: none

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The statistical evaluation of the number of live pups was performed. No statistically significant changes were recorded.
The total numbers of live pups and mean number of pups per litter (on the day of parturition/1st day after parturition and the 4th day of lactation) at all dose level was decreased in comparison with control group. At the dose level 1000 mg/kg/day decreased numbers of pups related with decreased numbers of females with live pups. At the dose levels 160 and 400 mg/kg/day the changes were not dose dependent.
Sex ratio of pups at all dose levels was balanced with the control. At the dose levels 400 mg/kg/day were detected increased portion of female pups than male pups.

Five pups from one litter at the dose level of 400 mg/kg/day and 12 pups - whole litter at the dose level of 1000 mg/kg/day were still born.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the litters at all dose levels were slightly lower compared to control but without dose dependence.
The mean body weights of the pups at all dose levels were in balance with control

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopical examination was performed in all pups. No pathological findings were recorded in pups of control group and treated groups.

Histopathological findings:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: none

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

not specified

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Table 3:

 

Mean body weight and body weight increment
Mean body weight (grams)					Mean body weight increment (grams/animal/day)
Application period	Dose level (mg/kg/day)				Application period	Dose level (mg/kg/day)
	0	160	400	1000		0	160	400	1000
Before application	341.12	341.95	342.49	342.11	Before application	-	-	-	-
1^st week	376.95	372.68	372.64	366.10	1^st week	5.12	4.39	4.31	3.43
2^nd week	394.83	399.00	396.58	389.71	2^nd week	2.55	3.76	3.42	3.37
3^rd week	413.71	416.98	413.06	403.09	3^rd week	2.70	2.57	2.35	1.91
4^th week	429.17	436.79	433.27	418.89	4^th week	2.21	2.83	2.89	2.26

Table 4:

Mean body weight and body weight increment (grams/animal/day)
Mean body weight						Mean body weight increment
Application period		Dose level (mg/kg/day)				Application period	Dose level (mg/kg/day)
		0	160	400	1000		0	160	400	1000
Before mating	Before application	229.00	228.94	228.83	228.42	Before application	-	-	-	-
	1^st week	234.92	235.79	236.23	234.31	1^st week	0.85	0.98	1.06	0.84
	2^nd week	241.31	244.77	245.42	243.06	2^nd week	0.91	1.28	1.31	1.25
		Mating period					Mating period
Day of pregnancy	0	246.70	249.61	250.95	253.36	0	-	-	-	-
	7	264.22	266.84	270.53	273.51	7	2.50	2.46	2.80	2.88
	14	298.62	295.93	306.48	303.69	14	4.91	4.16	5.14	4.31
	20	362.77	362.83	379.22	362.90	20	10.69	11.15	12.12	9.87
Day of lactation	0/1	286.18	304.51	304.02	301.00	0/1	-	-	-	-
	4	289.49	310.36	311.98	306.35	4	3.58	1.52	2.22	1.58

Table 5:

Mean food consumption per dose group (grams/animal/day)
Application period	Dose level (mg/kg/day)
	0	160	400	1000
1^st week	24.75	20.12	19.65	19.86
2^nd week	23.32	20.09	19.42	19.55
3^rd week	Mating period
4^th week	Mating period

Table 6: 

Mean food consumption per dose group (grams/animal/day)
Application period		Dose level (mg/kg/day)
		0	160	400	1000
Before mating	1^st week	15.25	13.87	11.64	13.72
	2^nd week	13.78	13.60	11.91	13.90
		Mating period
Day of pregnancy	7	11.67	11.38	11.25	11.11
	14	18.28	14.31*	15.10*	15.51*
	20	20.10	18.65	18.80	19.20
Day of lactation	0/1 - 4	26.33	19.64*	18.03*	18.79*

Table 7:

Mean weight of organs
Organs	Dose level (mg/kg/day)
	0		160		400		1000
	Absolute weight (g)	Relative weight	Absolute weight (g)	Relative weight	Absolute weight (g)	Relative weight	Absolute weight (g)	Relative weight
TESTES	3.5353	0.8235	3.4958	0.8022	3.6616	0.8475	3.6275	0.8742
EPIDIDYMIS	0.7380	0.1722	0.7728	0.1771	0.7579	0.1749	0.7137	0.1712
PROSTATE GLAND	0.8379	0.1948	0.8286	0.1900	0.8250	0.1902	0.7586	0.1810
PITUITARY GLAND	0.0127	0.0030	0.0117	0.0027	0.0134	0.0031	0.0111	0.0027

 

Table 8:

Mean weight of organs
Dose  (mg/kg/day)
	0		160		400		1000
Organs	Absolute weight (g)	Relative weight	Absolute weight (g)	Relative weight	Absolute weight (g)	Relative weight	Absolute weight (g)	Relative weight
OVARIES	0.1449	0.0486	0.1416	0.0455	0.1329	0.0429	0.1454	0.0476
UTERUS	0.9134	0.3058	0.8597	0.2772	0.9117	0.2922	0.9295	0.3006
PITUITARY GLAND	0.0188	0.0063	0.0167	0.0054	0.0189	0.0061	0.0179	0.0059

 

Table 9:

Macroscopic findings  (number of affected animals)
Pathological findings	Dose level (mg/kg/day)
	0	160	400	1000
Number of examined animals	12	12	12	12
Without macroscopic changes	11	12	0	0
Seminal vesicles: atrophy of left lobe	1	0	0	1
Liver: enlarged (1), light colour (2), marked structure (3), fattily (4)	0	0	(1)/9 (2)/5  (3)/11 (4)/1	(1)/12 (2)/1  (3)/11
Kidneys: enlarged (1), light colour (2), marked structure (3)	0	0	(3)/5	(1)/1 (2)/7 (3)/9

 

Table 10:

Macroscopic findings  (number of affected animals)
Pathological findings	Dose level (mg/kg/day)
	0	160	400	1000
Number of examined animals	12	12	12	12
Without macroscopic changes	12	11	7	1
Liver: enlarged (1), light colour (2), marked structure (3)	0	0	(1)/3 (3)/5	(1)/10 (2)/2 (3)/5
Vagina: blood in lumen	0	0	0	1
Ovaries: bursal cyst on right ovary	0	0	0	1
Uterus: dilatation	0	1	0	1
Mammary gland: nodule	0	0	0	1

 

Table 11:

Microscopical findings - MALES (number of affected animals)
Pathological findings	Dose level
	0	160	400	1000
Number of examined animals	12	0	12	12
Without microscopical changes	3	-	0	0
Pituitary gland: cyst in adenohypophysis	2	-	0	0
Testes:degeneration and/or atrophy of germinative epithelium (less than 10 % tubules)	3	-	-	1
Testes: exfoliation of germ cells to lumen of tubules (more than 10 %)	0	-	-	1
Testes: exfoliation of germ cells to lumen of tubules (to 10 %)	6	-	-	5
Testes: vacuolation of Sertolli cells	0	-	-	1
Epididymides – interstitium and epithelium - slight lymphocyte infiltration	0	-	-	2
Epididymides – germ cells in lumen of tubules	2	-	-	2
Prostate gland – atrophy of alveolar epithelium	0	-	-	1
Prostate gland – focal oedema of interstitium	1		-	1
Prostate gland - lymphocyte infiltration in interstitium	1	-	-	2
Seminal vesicles - focal desquamation of epithelium	0	-	-	1
Coagulating gland - focal desquamation of epithelium	0	-	-	1
Liver – hepatocellular hypertrophy	-	-	12	10
Liver – focal infiltration and/or fibrosis	-	-	2	3
Liver – focal vacuolation of hepatocytes	-	-	1	0
Kidneys – tubular hypertrophy in cortex	-	-	4	8
Kidneys – haemorrhage in cortex	-	-	0	1

Note: The reproductive organs were not examinated in males at the lowest and middle dose level groups. Microscopically examination of other organs was performed only in such dose level and for such organs where macroscopical findings organs were observed.

Table 12:

Microscopical findings - FEMALES (number of affected animals)
Pathological findings	Dose level (mg/kg/day)
	0	160	400	1000
Number of examined animals	12	12	12	12
Without microscopical changes	0	4	0	0
Liver: extramedullary hemapoiesis	-	-	3	3
Liver: hypertrophy	-	-	6	10
Mammary gland: myoepithelial adenoma	0	0	0	1
Uterus: focal accumulation of siderophages and lipophages in mesometrium	10	7	9	7
Uterus: siderophages in mucosa	7	1	8	5
Uterus: hydrometra	0	1	0	1
Uterus: acute endometritis	0	0	0	1
Uterus: organizing hematoma	3	3	1	3

Note: Microscopically examination of the liver was performed only in such dose level where macroscopically changed organs were observed

 

 

 

Applicant's summary and conclusion

Conclusions:

The growth (body weight and food consumption), clinical status and biometry and structure of reproductive organs of parental animals were not adversely affected by the test substance treatment. The quality of sperms of parental males was not adversely influenced by the 28th long test substance treatment.
Reproductive performance – ability of males to successfully mate was unaffected by the test substance treatment.
Reproductive performance of females - ability to produce viable offspring was affected by the test substance treatment.
The maternal toxicity (toxic effect on a pregnant woman or nursing mother) could affected an embryo and foetus.
Sex ratio and postnatal development of pups were not changed in treated groups but is not possible to exclude influence of the test substance treatment to prenatal development of offspring.

The test substance, 2- ETHYLHEXYLAL, directly affected the liver in males and females at the dose levels of 400 and 1000 mg/kg/day but this findings has no impact on establishing of NOAELs for reproduction and development.

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of males was established as 1000 mg/kg body weight/day. The conclusion is based predominantly on unaffected ability to successfully mate and unaffected quality of sperms.

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of females was established as 400 mg/kg body weight/day. Value based predominantly on decreased numbers of corpora lutea, decreased numbers of implantations and number of mothers with live born pups and increased number of the aborted females.

The NOAEL (No Observed Adverse Effect Level) for DEVELOPMENT of pups was established as 400 mg/kg body weight/day. Value based predominantly on the major manifestations of developmental toxicity death of the developing organism. The toxic effect of the test substance on the structurnal abnormality, altered growth and functional dificiency is not possible to exclude due to abortions and cannibalism of the developing organism.

Executive summary:

Introduction

The test substance, 2-ETHYLHEXYLAL, was tested for reproduction toxicity using the OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on July 27^th 1995.

This study provides information on the possible harmful effects of the test substance on reproduction and development after repeated exposure on parental generation and first generation pups.

 

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered in olive oil (pharmaceutical quality) using a stomach tube; oral application of rats was made daily. The concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. Four groups of animals were included in the study - 3 treated groups (doses 160, 400, 1000 mg/kg of body weight/day) and one control group (vehicle only). Each group consisted of 12 males and 12 females. The dose levels for study were chosen on the basis of the results of the dose-range finding experiment with 14-day application period.

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females  – during pregnancy and till the 3^rd day of lactation,

males  –  after mating period – totally for 28 days,

non pregnant females (mated females without parturition) – for 25 days after the confirmed mating.

During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in specified time intervals. Vaginal smears were prepared daily during mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) were also recorded.

The study was finished by gross necropsy of animals. In all males of all groups the sperm parameters: sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

 

Results

The test substance, 2- ETHYLHEXYLAL, did not seriously affect growth of parental males and females at all dose levels. The results relevant to mean body weight males and females were not statistically significant.

The slightly lower food consumption was recorded in treated males.  The mean body weight increments of treated mothers in lactation period at all dose levels (not dose dependent) were decreased against control, which related with decrease mean xx consumption of treated mothers in pregnancy and lactation (statistically significant at the 2^nd week of pregnancy and in lactation). The results relevant to growth could be considered not to be of toxicological importance.

 

The clinical examination revealed congestion of mucous membranes and skin of apical part of the body in all treated animals since the 2^nd week. The macroscopic changes in the liver (enlarged, light colour, marked structure) were observed in males and females at the dose levels of 400 and 1000 mg/kg/day. The changes in the kidneys (enlarged, light colour, marked structure) were detected only in males at the dose levels of 400 and 1000 mg/kg/day.

In females of the highest dose the salivation (only in 2 females) and piloerection (only in one female) were observed sporadically.

 

The biometry of organs showed only statistically insignificant differences in treated parental males and females. Decrease of absolute and relative weight of prostate gland was recorded in parental males of the highest dose level and was not considered to be toxicologically relevant because microscopic findings (focal oedema and lymphocyte infiltration in interstitium) were observed also in control males. In males at the dose level of 1000 mg/kg/day was also observed reduction of absolute and relative weight of epididymis and pituitary gland. Changes in these organ weights were not accompanied by significant morphological changes in the same tissues.

The histopathological findings in epididymis (slight lymphocyte infiltration of the interstitium and epithelium) were observed in two males at the highest dose. The finding of germ cells in lumen of tubules (in two males at the highest dose) is very sensitive indicator of spermatogenic disturbance in the rat but the same number of affected animals was observed also in control animals.

 

The histopathological examination of the liver revealed findings in animals of the doses 400 and 1000 mg/kg/day (hypertrophy, in some females accompanied by extramedullary hemopoiesis; focal infiltration and vacuolation in males) caused by the test substance. It is probably adaptation response to application of the test substance In male kidneys at the middle and high dose the tubular hypertrophy and in one case haemorrhage in cortex were observed.

 

The relative and absolute weights of organs in treated females were not statistically significant different versus control animals and histopathological examination of female reproductive organs and pituitary gland did not reveal any serious changes. Only changes relating to previous gravidity or proceeded oestrous cycle were recorded.

 

Absolute and relative weights of the testes were not changed in treated males and were similar to control. Histopathological examination of the testes of parental males showed some changes of testicular microscopical structure but without damage of spermiogenesis. The exfoliation of germ cells to lumen of tubules was observed in treated and also in control animals so it can not be assessed as spermatogenic disturbance factor which was caused by the test substance. 

 

Observation of sperm motility and sperm morphology did not reveal serious damage of sperms quality in treated males versus control. The test substance did not influence sperm motility. The percentual proportion of morphologically changed sperms was slightly increased in all treated males. The most frequent changes were bent tails in sperms. In spite of sporadic change in sperm morphology the fertility of treated males was not affected. But the animals in the reproduction screening study are treated for the time period which is shorter than the duration of whole spermatogenic cycle so that an effect on spermatogenesis may not have had adequate time to become evident as reduced sperm counts that affect fertility.

 

Observation of pups revealed statistically insignificant difference. The mean body weights of pups on the day of parturition/1^st day after parturition and the 4^th day of lactation were comparable. Sex ratio were unaffected by the test substance treatment.

The litter size (an important indicator of overall reproduction performance) was changed – decreased number of pups at all dose levels versus control group was observed. Marked decreasing of mean number of pups per litter in the highest dose level was probably caused by decreased numbers of mothers delivered live pups (11-9-10-7). The parturition of stillborn pups was observed at the dose levels of 400 and 1000 mg/kg/day. Mortality of live born pups was not detected.

 

Reproduction parameters – ability of males and females to successfully mate and achieve pregnancy was not adversely influenced by the test substance treatment. The number of females achieving pregnancy and duration of pregnancy were unchanged in treated groups compared to control. The duration of mating was slightly longer at the highest dose level.

The fertility index was decreased without dose dependence and the alteration was observed also in control group. In fertility index may be difficult to determinate the affected sex when both sexes were dosed furthermore males were dosed for less than the duration of the spermatogenic cycle so reproductive effect may not be manifested in the fertility index.

The mating index and gestation index were decreased at the highest dose level. The numbers of aborted females were increased with doses. At the highest dose level 2 aborted females, at the middle dose level 1 aborted female were observed.

The decreased numbers of corpora lutea and numbers of implantations at the highest dose level manifested statistically insignificant differences. Slight decreased of pre-implantation losses, post-implantation losses and post-natal losses at the highest dose level associated with lower number of corpora lutea, implantations and number of live pups.

The survival index – an important endpoint reflects the ability of the pups to survive – was not changed.

   

Conclusion

The growth (body weight and food consumption), clinical status and biometry and structure of reproductive organs of parental animals were not adversely affected by the test substance treatment. The quality of sperms of parental males was not adversely influenced by the 28^th long test substance treatment.     

Reproductive performance – ability of males to successfully mate was unaffected by the test substance treatment.

Reproductive performance of females - ability to produce viable offspring was affected by the test substance treatment.

The maternal toxicity (toxic effect on a pregnant woman or nursing mother) could affect an embryo and foetus.

Sex ratio and postnatal development of pups were not changed in treated groups but is not possible to exclude influence of the test substance treatment to prenatal development of offspring. 

 

The test substance, 2- ETHYLHEXYLAL, directly affected the liver in males and females at the dose levels of 400 and 1000 mg/kg/day but this findings has no impact on establishing of NOAELs for reproduction and development.

 

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of males was established as 1000 mg/kg body weight/day. The conclusion is based predominantly on unaffected ability to successfully mate and unaffected quality of sperms.

 

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of females was established as 400 mg/kg body weight/day. Value based  predominantly on decreased numbers of corpora lutea, decreased numbers of implantations and number of mothers with live born pups and increased number of the aborted females. 

    

The NOAEL (No Observed Adverse Effect Level) for DEVELOPMENT of pups was established as 400 mg/kg body weight/day. Value based predominantly on the major manifestations of developmental toxicity death of the developing organism. The toxic effect of the test substance  on the structurnal abnormality, altered growth and functional dificiency is not possible to exlude due to abortions and cannibalism of the developing organism.