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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Experimental result published in peer reveiwed journal. Some details on methods / results not available

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Diethyl phthalate
EC Number:
201-550-6
EC Name:
Diethyl phthalate
Cas Number:
84-66-2
Molecular formula:
C12H14O4
IUPAC Name:
1,2-diethyl benzene-1,2-dicarboxylate
Details on test material:
- Name of test material (as cited in study report): diethyl phthalate
- Physical state: liquid
- Analytical purity: 99.8%
- Lot/batch No.: SEM5441

Test animals

Species:
rat
Strain:
other: Crj:CD (SD) IGS
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Tsukuba breeding Center Charles River japan Inc
- Age at study initiation: (P) x wks; (F1) x wks: P 4 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no
- Housing: individually except during acclimatisation (1 or 2 animals same sex)
- Use of restrainers for preventing ingestion (if dermal): yes/no: N/A
- Diet (e.g. ad libitum): NIH-07M Clea Japan Inc Tokyo Japan
- Water (e.g. ad libitum): tap water
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/- 3degrees C
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: basal diet
Details on exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): basal NIH-07M diet

VEHICLE
- Justification for use and choice of vehicle (if other than water): dietary study; in addition DEP is poorly soluble in water
- Concentration in vehicle: 600, 3000 & 5000 ppm
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until the day of sucessful copulation
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: vaginal plug/sperm; day 0
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Not reported
- Further matings after two unsuccessful attempts: [no / yes (explain)]: procedure repeated until pregnancy resulted for 3 week mating period
- After successful mating each pregnant female was caged (how): singly
- Any other deviations from standard protocol: not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Not reported
Duration of treatment / exposure:
Approximately 15 weeks for male and 17 weeks for female parents of the F0 & F1 generations.
Frequency of treatment:
Continuously via the diet
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21-25 days of age.
- Age at mating of the mated animals in the study: 15 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control) 600, 3000 & 15000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: selected on basis of results of a preliminary study
- Rationale for animal assignment (if not random): assigned on the basis of weight so that the 4 groups had similar group mean and SD values
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for clinical signs & mortality

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:Not reported

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: Yes
Oestrous cyclicity (parental animals):
yes,see table below
Sperm parameters (parental animals):
Sperm parameters were measured in all F0 & F1 males surviving to scheduled terminal kill. The right cauda epididymis was weighed & used for sperm analysis. Right testis was also used used for counting homogenisation resistant testicular spermatids.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:] seee table below

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.] yes gross examination
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]: after weaning of their F1 & F2 offspring as appropriate
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]: after weaning of their F1 & F2 offspring as appropriate

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]: yes

HISTOPATHOLOGY / ORGAN WEIGHTS
Selected tissues were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [26] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: macroscopic & microscopic examination

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]: yes

HISTOPATHOLOGY / ORGAN WEIGTHS
Selected tissues were prepared for microscopic examination and weighed, respectively.For control & 15000 ppm groups all F0 & F1 parental animals were examined for abnormalities of the reproductive organs. In the low dose group the same organs were examined in female animals with abnormal oestrus cycles, males with sperm abnormality & pairs of animals that failed to mate or produce offspring. In addition the kidneys of F1 parental females in control & 15000 groups were examined due to the significant increase in weight noted when compared with controls.
In F1& F2 weanlings the thymus of 6 of each of the males & females & the spleen of 6 F2 males in the control & 15000 ppm groups were examined as the weights were significantly decreased in the 15000 group compared with controls.
Statistics:
For body weights, body weight gain, food consumption, oestrus cyle length, number of implants & pups delivered, delivery index sperm parameters ormone levels, absolute & relative organ weights, time of reflex response completion, pinna detachment, eye opening, incisor eruption, age & body weights at sexual maturation, AGD & and pup viability homogeneity of variance was evaluated using Bartlett's test then if homogeneous one way analysis of variance was used to determine if there were statistical differences between the groups and their counterparts in the controls.If the analysis of variance gave a significant result , Dunnetts test was performed to detect the differences between controls & the DEP groups.
When Bartlett's test detected non homogeneous results Kruskal-Wallis test was used to detect significant differences and then the Mann Whitney U test was performed to detect any significant differences between the DEP groups and the corresponding control group.
Statistical aanalyses was performed using litter means for body weights and the AGDs of pups /sex before weaning.
The completion of physical development of pups was assessed by the Wilcoxon rank test on the litter data,
The Chi squared /Fishers exact probability tests were used to analyse incidence of females with normal oestrus cycles & indices of copulation, fertility & gestation, & sex ratios of pups.
Reproductive indices:
See table below
Offspring viability indices:
See table below

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Incidences of abnormal sperm & tailess spermwere significantly increased in males of F0 generation given 3000 ppm DEP and in males of the F1 generation given 3000 and 15000 ppm.
Reproductive performance:
no effects observed

Details on results (P0)

N/A as no adverse effects were observed on systemic & reproductive parameters at dietary levels of up to 15000 ppm.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed on systemic & reproductive parameters at dietary levels of up to 15000 ppm.
Remarks on result:
other: Generation: F0 & F1 in parents (migrated information)
Dose descriptor:
NOAEL
Effect level:
3 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: In terms of development and growth pup body weight gain before weaning was reduced in the 15000 ppm group.
Remarks on result:
other: Generation: F1 & F2 pups (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Description (incidence and severity):
examination limited to thymus & spleen

Details on results (F1)

N/A as the effect on development and growth was reduced pup body weight gain before weaning in the 15000 ppm group

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Reproductive data for F0 and F1 parental animals

 

DEP (ppm) Administered

 

0 (control)

600

3000

15000

F0 parental animals

 

 

 

 

No. of pairs

24

24

24

24

Precoital interval (days)

3.4±3.8a

2.7±1.5

3.7±3.5

2.4±1.2

Copulation index (%)b

 

 

 

 

Male

95.8

95.8

100

100

Female

100

95.8

100

100

Fertility index (%)c

 

 

 

 

Male

95.7

95.7

100

95.8

Female

95.8

95.7

100

95.8

Gestation index (%)d

95.7

100

100

95.7

Gestation length (days)

22.3±0.5

22.3±0.5

22.3±0.5

22.1±0.3

No. of implantations

14.7±1.8

15.0±1.7

14.9±2.0

14.8±2.1

Delivery index (%)e

94.3±6.7

91.3±8.8

93.8±5.0

94.9±7.2

No. of pups delivered

13.9±2.0

13.7±2.0

14.0±2.1

14.0±2.2

F1 parental animals

 

 

 

 

No. of pairs

24

24

24

24

Precoital interval (days)

2.5±1.6

2.8±1.2

4.3±4.7

3.8±3.8

Copulation index (%)

 

 

 

 

Male

100

100

87.5

95.8

Female

100

100

95.8

100

Fertility index (%)

 

 

 

 

Male

95.8

95.8

95.2

95.7

Female

95.8

95.8

95.7

95.8

Gestation index (%)

100

100

100

100

Gestation length (days)

22.4±0.5

22.3±0.5

22.3±0.6

22.1±0.3*

No. of implantations

14.3±2.5

14.3±3.2

14.9±2.7

14.7±2.5

Delivery index (%)

91.3±11.1

93.7±8.4

91.8±13.2

98.8±17.9

No. of pups delivered

13.0±2.8

13.5±3.5

13.9±3.1

13.3±3.5

a Mean±SD; b Copulation index (%) = (No. of rats with successful copulation/no. of rats paired) x100; cFertility index (%) = (No. of females pregnant or no. of males sired/no. of rats with successful copulation) x100; d Gestation index (%) = (No. of females with parturition/no. of females pregnant) x100; e Delivery index (%) = (No. of pups delivered/no. of implantations) x100; *               Significantly different from the control, p0.05

Litter data on reproductive parameters for F1 and F2 offspring

 

DEP (ppm)

 

0 (control)

600

3000

15000

F1 offspring

 

 

 

 

No. of litters examined

22

22

24

22

Sex ratio of pupsb

0.544

0.465

0.507

0.490

Viability index before weaning (%)cde

 

 

 

 

Day 0

97.6

98.8

97.8

97.2

Day 4

97.8

96.2

97.2

97.7

Day 21

97.7

95.5

90.6

88.1

Male pup weight before weaning (g)

 

 

 

 

Day 0

6.9±0.7a

7.0±0.6

7.0±0.7

6.7±0.7

Day 4

10.6±1.4

10.1±1.4

10.5±1.9

9.7±1.4

Day 21

59.0±7.7

56.8±6.6

55.1±9.5

48.1±4.8f**

Female pup weight before weaning (g)

 

 

 

 

Day 0

6.5±0.7

6.5±0.7

6.5±0.7

6.3 b±0.6

Day 4

10.2±1.4

9.6±1.5

9.9±1.6

9.1±1.1*

Day 21

56.3±6.6

55.4±7.0

53.8±7.2

45.8±4.5f**

Anogenital distance (mm)

 

 

 

 

Male Day 0

3.67±0.31

3.72±0.29

3.83±0.35

3.56±0.28

Day 4

5.66±0.50

5.46±0.44

5.54±0.69

5.54±0.55

Female Day 0

1.76±0.21

1.67±0.22

1.67±0.23

1.77±0.22

Day 4

2.77±0.34

2.70±0.25

2.72±0.34

2.74±0.33

F2 offspring

 

 

 

 

No. of litters examined

23

23

22

23

Sex ratio of pups

0.510

0.510

0.523

0.482

Viability index before weaning (%)

 

 

 

 

Day 0

96.2

98.6

97.9

98.6

Day 4

96.3

97.8

99.4

98.1

Day 21

94.0

96.7

98.9

99.5

Male pup weight before weaning (g)

 

 

 

 

Day 0

7.1±0.5

7.0±0.7

6.9±0.7

6.8±0.6

Day 4

11.4±1.8

10.6±1.5

10.7±1.2

10.8±1.6

Day 21

60.9±9.5

61.4±6.9

60.6±4.5

53.8±4.8**

Female pup weight before weaning (g)

 

 

 

 

Day 0

6.6±0.6

6.5±0.7

6.4±0.5f

6.3±0.4g

Day 4

10.8±2.1

9.9±1.4

10.0±0.9f

10.0±1.1g

Day 21

58.2±9.4

58.7±6.2

58.2±4.2f

51.5±4.2g **

Anogenital distance (mm)

 

 

 

 

Male Day 0

3.63±0.29

3.59±0.17

3.64±0.20

3.65±0.24

Day 4

5.78±0.42

5.66±0.39

5.73±0.36

5.77±0.49

Female Day 0

1.76±0.11

1.73±0.09

1.76±0.09f

1.74±0.09g

Day 4

2.93±0.23

2.89±0.22

2.90±0.21f

2.88±0.20g

a Mean±SD; b Sex ratio of pups = No. of male pups/total no. of pups; c Viability index on postnatal day 0 (%) = (No. of live pups delivered/total no. of pups delivered) x100

d Viability index on postnatal day 4 (%) = (No. of live pups on postnatal day 4/no. of live pups delivered) x100 ; e Viability index on postnatal day 21 (%) = (No. of live pups on postnatal day 21/no. of live pups on postnatal day 4 after cull) x100; f and g Values obtained from 21 and 22 litters, respectively; * Significantly different from the control, p0.05; ** Significantly different from the control, p0.01

Hepatic microsomal cytochrome P450 isozyme contents in F0 parental males

 

DEP (ppm)

 

0 (control)

600

3000

15000

No. of F0 parental males examined

6

6

6

6

CYP3A2 (p mol/mg protein)

55.6±20.5

62.2±18.5

33.2±13.1

92.0±15.4**

CYP4A1 (p mol/mg protein)

13.7±3.1

15.0±6.9

11.5±2.6

62.8±19.2*

a              Mean±SD; *            Significantly different from the control, p0.05; **          Significantly different from the control, p0.01

Serum hormone levels in F0 parental males

 

DEP (ppm)

 

0 (control)

600

3000

15000

No. of F0 parental males examined

6

6

6

6

Testosterone (ng/mL)

2.63±1.23a

1.81±0.46

0.50±0.26**

1.26±0.66*

Progesterone (ng/mL)

2.66±2.51

3.61±2.50

5.98±3.00

3.08±0.94

a              Significantly different from the control, p0.05;             **            Significantly different from the control, p0.01

Applicant's summary and conclusion

Conclusions:
Following dietary administration to rats through 2 successive generations, the NOAEL for general toxicity and reproductive performance in parental animals is considered to be 15000 ppm as there were no adverse effects on these parameters.
For development and growth of pups the NOAEL is considered to be 3000 ppm due to decreased body weight gain in those given 15000 ppm.
Executive summary:

Reproductive toxicity has been investigated in a 2 -generation study in rats using methods in accordance with OECD test methods. The the NOAEL for general toxicity and reproductive performance in parental animals was considered to be 15000 ppm (equivalent to a mean intake of 1016 -1297 mg/kg/day) as there were no adverse effects on these parameters. For development and growth of pups the NOAEL was considered to be 3000 ppm (equivalent to a mean intake of 222 -267 mg/kg/day) due to decreased body weight gain in those given 15000 ppm (equivalent to a mean intake of 1150 -1375 mg/kg/day).