Alkanes, C14-20

Toxicity to birds

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study does not strictly follow the guideline recommendations and does not give results for all the endpoints (no 14-d survival of hatchlings followed). However, the results are numerous and consistent.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.2300 (Avian Reproduction Test)

Deviations:

yes

Remarks:

Only 1 concentration tested; 8 pens instead of 12; longer final phase; no 14-d survival of hatchlings observed

Principles of method if other than guideline:

Guideline principles

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No test surrogate material

Dose method:

other: Food available ad libitum

Analytical monitoring:

not specified

Vehicle:

no

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: yes
- Food composition: Food consisted of duck developer mash (not less than 14.5% crude protein, 2.75% crude fat, and 7% crude fiber) for 2 months and then duck breeder mash (not less than 17% crude protein, 3% crude fat, and 6% crude fiber).

- Nominal concentration : 1.0% of paraffin mixture

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard duck
- Source: Commercial
- Age at test initiation (mean and range, SD): 5 months
- Weight at test initiation (mean and range, SD): No data
- Sexes used: Males and females

Limit test:

yes

Total exposure duration (if not single dose):

26 wk

Post exposure observation period:

No post-exposure observation period

No. of animals per sex per dose and/or stage:

8

Control animals:

yes, concurrent no treatment

Nominal and measured doses / concentrations:

Nominal concentration : 1.0% parffin mixture in food

Details on test conditions:

ACCLIMATION
- Acclimation period: 2 weeks
- Acclimation conditions (same as test or not): same as test
- Feeding: Duck developper mash (not less tahn 14.5% crude protein, 2.75% crude fat, and 7% crude fiber), ad libitum
- Health (any disease or mortality observed): No data
- Fasting period before study: No fasting period


PEN SIZE AND CONSTRUCTION MATERIALS
- Description: 90 x 90x 60-cm vinyl coated wire pens
- Floor covering: No data
- Caging: pairs

NO. OF BIRDS PER REPLICATE
- For negative control: 1 male / 1 female
- For treated: 1 male / 1 female

NUMBER OF REPLICATES
- For negative control: 8
- For treated: 8

TEST CONDITIONS (range, mean, SD as applicable)
- Photoperiod: Artificial day length was set at 10 hrs for the first 2 months, then increased by 2 hrs at weekly intervals to 16 hrs where it was maintained.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations:
- Remarks:


BODY WEIGHT
- Time schedule for examinations: Monthly
- Remarks:


FOOD CONSUMPTION (if feeding study)
- Time schedule for examinations:No data
- Remarks:


PATHOLOGY
All birds were sacrificed after 26 weeks on treatement. Protions of the kidney, liver, and gonad were preserved in 10% formalin and later embedded in paraffin, sectioned and stain with hematoxylin-eosin for microscopic observation.


ORGAN WEIGHTS
- Organs: Liver, spleen, adrenal gland and gonad (oviduct or testes)
- Remarks: organs were weight at the end of the experiment after a 26-week exposure


OTHER:
At each weighing (monthly intervals), 3-ml blood samples were drawn from the alar vein for haematological (hematocrit and hemoglobin determinations) and biochemical (plasma concentrations of sodium, potassium, cholesterol, and triglycerides; plasma enzyme activities: glutamic oxalacetic transaminase- GOT, lactate deshydrogenase - LDH, alpha-hydroxybutiric acid deshydrogenase - HBDH, ornithine carbamyl transferase - OCT) investigations.
Portions of selected tissues were frozen for petroleum hydrocarbon analyses.

Details on reproductive parameters:

The following parameters were examined per parental pen per week:
- Eggs laid
- Eggshell thickness or eggshell strength
- Eggs fertile
- Embryos viable
- Normal hatchlings
- Abnormal hatchlings


OTHER:
Content of the eggs were saved for later petroleum hydrocarbon analyses.

Reference substance (positive control):

no

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

body weight

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

organ weights

Remarks:

Liver

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

organ weights

Remarks:

Spleen

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

organ weights

Remarks:

Adrenal gland

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

organ weights

Remarks:

Gonads (ovary or testis)

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

reproductive parameters

Remarks:

Eggs laid

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

reproductive parameters

Remarks:

Fertility

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

reproductive parameters

Remarks:

Hatchability

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

reproductive parameters

Remarks:

Eggshell thickness

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

pathology

Remarks:

Liver histopathology

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

pathology

Remarks:

Kidney histopathology

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

pathology

Remarks:

Gonad (testis or ovary) histopathology

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

pathology

Remarks:

Blood hematocrit and hemoglobin concentration

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

pathology

Remarks:

Plasma sodium and potassium concentrations

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

pathology

Remarks:

Plasma triglyceride and cholesterol concentrations

Duration (if not single dose):

26 wk

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

pathology

Remarks:

Plasma enzyme activity: GOT, HBDH and OCT

Repellency factors (if applicable):

No data

Mortality and sub-lethal effects:

For details see Tables no. 1 and 2
MORTALITY
No mortality observed

CLINICAL SIGNS
- Results: No difference between exposed and control birds


ABNORMAL BEHAVIOUR
- Results: No abnormal behaviour


BODY WEIGHT (see Table 1)
- Results: No difference between exposed and control birds


FOOD CONSUMPTION (if feeding study)
- Results: No difference between exposed and control birds


PATHOLOGY
- Results: No difference between exposed and control birds

ORGAN WEIGHTS
- Results: No difference between exposed and control birds


HISTOPATHOLOGY
- Results: No difference between exposed and control birds

Effects on reproduction:

See table 2

Results with reference substance (positive control):

No reference substance

Further details on results:

When activities of plasma enzymes originating from the liver (GOT), Kidney (OCT), or heart (HBDH) were compared between treated and control birds, there were no obvious differences due to treatment. In addition plasma glyceride and cholesterol concentrations did not change with treatment, nor did variables that can be altered by dehydration, e.g., hematocrit, hemoglobin, sodium, potassium and total protein in the plasma.

Reported statistics and error estimates:

According to the statistical test used for evaluating significance of the results, there was no statistical difference between treated and control birds for any of the parameters analysed (p<0.05).

Table 1. Weights of whole bodies and organs of adult Mallard ducks after 26 weeks on diet contaminated with paraffin mixture

 

Treatment	Sex	Body weight (g)	Organ weights (g/1000g body weight)
			Liver	Spleen	Gonad
Control	Males	1339 ± 66.8	22.1 ± 0.86	0.49 ± 0.04	26.0 ± 4.16
	Females	1274 ± 26.3	41.5 ± 3.5	0.66 ± 0.06	16.6 ± 4.86
1.0% Paraffin	Males	1268 ± 69.8	21.7 ± 0.91	0.47 ± 0.05	24.3 ± 4.09
	Females	1186 ± 40.9	43.6 ±5.44	0.56 ± 0.06	25.2 ± 5.2

 

 

 

Table 2. Egg production in adult Mallard ducks after 26 weeks on diet contaminated with paraffin mixture

 

Treatment	Number of total eggs laid	Number of Eggs laid / hen	Number of Eggs laid / hen / week
Control	615	76.9	5.1
1.0% paraffin	617	77.1	5.1

 

Conclusions:

No long-term toxicity of paraffin was observed neither to adult Mallard ducks, neither to their eggs.

Executive summary:

Adult Mallard ducks were exposed to diet contaminated with 1.0% paraffin mixture (equal parts of tridecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, 2,2,4,6,6-pentamethylheptane, 2,2,4,4,6,8,8-heptamethylnonane, and decahydronaphthalene) for 26 weeks. No statistical differences were seen between treated and control duck body weight, organ (liver, kidney, spleen) weights, blood parameters (hematocrit, hemoglobin, plasma sodium, potassium, cholesterol and triglycerides, plasma enzyme activities glutamic oxalacetic transaminase - GOT, lactated deshydrogenase - LDH, alpha-hydroxybutiric acid dehydrogenase - HBDH, and orinithine carbamyl transferase - OCT). The exposure had no effect on the number of laid eggs, neither on eggshell thickness, fertility and egg viability.

Reference 2

Endpoint:

long-term toxicity to birds

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study does not follow a guideline, but it is reliable because the results are numerous and consistent.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

The ducks are fed on a commercial breeder mash containing the test substance for 7 months. They are weighed monthly and blood is taken up from the brachial vein to follow liver function using indocyanin green dye clearance technique (Ketterrer, S. G., Wiegand, B. D., Rapaport, E., 1960. Hepatic uptake and biliary excretion of indocyanin green and its use in estimation of hepatic blood flow in dogs. American Journal of Physiology 199: 481-484).

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No test surrogate material

Dose method:

feed

Analytical monitoring:

not specified

Vehicle:

no

Details on preparation and analysis of diet:

Commercial breeeder mash containing 1% of the paraffin mixture

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard duck
- Source: no data
- Age at test initiation (mean and range, SD): no data
- Weight at test initiation (mean and range, SD): 1200-1300 g
- Sexes used: Males

Limit test:

yes

Total exposure duration (if not single dose):

7 mo

Post exposure observation period:

No post exposure period

No. of animals per sex per dose and/or stage:

12

Control animals:

yes, concurrent no treatment

Nominal and measured doses / concentrations:

Nominal concentration in food: 1 %

Details on test conditions:

ACCLIMATION
- Acclimation period: 1 month
- Acclimation conditions (same as test or not): Same as test
- Feeding: commercial breeder mash ad libitum
- Health (any disease or mortality observed): no data
- Fasting period before study: no fasting period


PEN SIZE AND CONSTRUCTION MATERIALS
- Description: vinyl coated wire pens, 90 x 90 x 60 cm
- Floor covering: no data
- Caging: pairs
- air-conditioned building with controlled lighting and running water

NO. OF BIRDS PER REPLICATE
- For negative control: 2
- For treated: 2


NO. OF REPLICATES PER GROUP
- For negative control: 6
- For treated: 6


TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: no data but controlled (air-conditioned)
- Photoperiod: 12D:12L

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Monthly


BODY WEIGHT
- Time schedule for examinations: monthly


PATHOLOGY
- ICG test performed monthly
- Blood parameters (Plasma sodium and potassium, glutamic oxaloacetic transaminase - GOT): monthly


ORGAN WEIGHTS
- Organs: Liver, testes

Details on reproductive parameters:

No reproductive parameters studied

Reference substance (positive control):

no

Duration (if not single dose):

7 mo

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

body weight

Duration (if not single dose):

7 mo

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

other: ICG test

Duration (if not single dose):

7 mo

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

organ weights

Remarks:

Liver

Duration (if not single dose):

7 mo

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

organ weights

Remarks:

testes

Duration (if not single dose):

7 mo

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

pathology

Remarks:

Blood hematocrit

Duration (if not single dose):

7 mo

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

pathology

Remarks:

Plasma sodium and potassium

Duration (if not single dose):

7 mo

Dose descriptor:

other: NOELR

Effect level:

10 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

pathology

Remarks:

Plasma GOT

Repellency factors (if applicable):

Not applicable

Mortality and sub-lethal effects:

MORTALITY
No mortality


ABNORMAL BEHAVIOUR
- Results: No abnormal behaviour


BODY WEIGHT
- Results: No statistical difference between control and treated duck body weights (p<0.05)


PATHOLOGY
- Results: There was no statistical difference between control and treated duck for any of the investigated parameters


ORGAN WEIGHTS
- Results: see table 1

Effects on reproduction:

Not applicable

Results with reference substance (positive control):

Not applicable

Further details on results:

No further details

Reported statistics and error estimates:

The data were analyzed using a two-way analysis of variance for repeated measures. Separation of mean differences was performed using Duncan's multiple range test.

Table 1. Mean liver and testes weights (± SE) in groups of 12 ducks fed paraffinic petroleum hydrocarbons for 7 months

 

Treatment	Liver		Testes
	Total (g)	g/kg body weight	Total (g)	g/kg body weight
Control	23.8 ± 1.9	17.5 ± 1.4	14.7 ± 2.1	11.0 ± 1.5
Paraffin	25.4 ± 1.8	18.9 ± 1.2	20.4 ± 3.3	15.7 ± 3.0

 

Validity criteria fulfilled:

not applicable

Conclusions:

The paraffin mixture is not toxic to Mallard duck.

Executive summary:

The indocyanine green dye clearance test (ICG) for hepatic function was determined in mallard ducks before and during the chronic ingestion (7 months) of representative paraffinic petroleum hydrocarbon mixture (tridecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, 1,1,4,6,6-pentamethylheptane, 2,2,4,4,6,8,8-heptamethylnonane, 2,6,10,14-tetramethylpentadecane, decahydronaphthalene).

No mortality nor visible symptoms of toxicity occurred in any of the tests. There was no statiticaly significant difference between control and treated duck weights. There was no change in ICG, nor in plasma sodium, potassium and GOT activity. It is concluded that paraffin mixture is not toxic to Mallard duck.

Reference 3

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No guideline. The effects on embryonic development are investigated through direct application of the substance on the egg shell. A great number of eggs (65 per dose) were tested, which contributes to make the results statistically valid.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Oil is applied on the surface of fertile eggs. Embryonic survival time, teratogenic potential and growth are evaluated.

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No surrogate test material

Dose method:

other: application onto egg shell

Analytical monitoring:

no

Vehicle:

no

Details on preparation and analysis of diet:

No diet

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard duck
- Source: Commercial hatcheries: Whistling WIngs, Hanover, Ill., and Truslow Farms, Chestertown, Md.
- Age at test initiation (mean and range, SD): Eggs (72hr of development)

Limit test:

no

Total exposure duration (if not single dose):

18 d

Post exposure observation period:

No post-exposure period

No. of animals per sex per dose and/or stage:

65 eggs per dose. For sex ratio, see results.

Control animals:

yes, concurrent no treatment

Nominal and measured doses / concentrations:

5µL per egg

Details on test conditions:

SUBSTANCE APPLICATION:
5µL of paraffin mixture were applied by microliter pipet to the shell surface just below the air space of upright eggs (72 hrs of development) and permitted to spread freely


NO. OF EGGS
- For negative control: 65
- For treated: 65


TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: 37.5°C
- Relative humidity (%): 70

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: daily
- Remarks: all eggs were candled daily to determine mortality, and dead embryos were removed and examined.


EMBRYONIC WEIGHT
- Time schedule for examinations: All remaining eggs were opened on the 18th day of development and the embryos were removed and weighed.
- Remarks:


PATHOLOGY
- One half of the embryos were defeathered and placed in Bouin's solution for fixation and subsequent examination of soft tissues by the sectioning technique of Wilson (1965) and by dissection.
- The other half were cleared and staiend with alizarine red S according to the method described by Karnofsky (1965) for squeletal examination.



OTHER:
When removed from the shell, sex-ratios, crown-rump lengths, and beak lengths were recorded.

Details on reproductive parameters:

The following parameters were examined per egg:
- Embryos viable
- Clinical signs of toxicity, abnormalities and mortality


OTHER:
- Embryonic growth: embryo weight, crown-rump length, beak length
- Sex ratio

Reference substance (positive control):

no

Duration (if not single dose):

18 d

Dose descriptor:

other: NOELR

Effect level:

5 other: µL/egg

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Remarks:

Embryonic mortality

Duration (if not single dose):

18 d

Dose descriptor:

other: NOELR

Effect level:

5 other: µL/egg

Conc. / dose based on:

test mat.

Basis for effect:

reproductive parameters

Remarks:

Embryo weight

Duration (if not single dose):

18 d

Dose descriptor:

other: NOELR

Effect level:

5 other: µL/egg

Conc. / dose based on:

test mat.

Basis for effect:

reproductive parameters

Remarks:

Sex ratio

Duration (if not single dose):

18 d

Dose descriptor:

other: NOELR

Effect level:

5 other: µL/egg

Conc. / dose based on:

test mat.

Basis for effect:

other: Development: crown-rump length

Duration (if not single dose):

18 d

Dose descriptor:

other: NOELR

Effect level:

5 other: µL/egg

Conc. / dose based on:

test mat.

Basis for effect:

other: Development: beak length

Repellency factors (if applicable):

Not applicable

Mortality and sub-lethal effects:

For details on mortality and sub-lethal effects see Table no.1.

Effects on reproduction:

Effect on embryo development : see table1

Results with reference substance (positive control):

No reference substance

Further details on results:

No other details

Reported statistics and error estimates:

Embryonic weights, crown-rump lengths, and beak lengths were compared by one-way analysis of variance and Duncan's multiple range test. Survival data, sex distribution and numbers of embryos with one or more defects were compared by squared-chi analysis.
No statistical difference ws found between treated and control eggs for any invetigated parameter.

Table 1. Effects of paraffin mixture (5 µL/egg) on Mallard duck embryos

 

	Control	Paraffin
Number treated	65	65
Percentage survival (Days 3-18)	97	94
Sex ratio, M:F	45:55	44:56
Embryonic Weight (g)
Male	15.72 ± 1.78	16.34 ± 1.49
Female	15.75 ± 2.03	15.98 ± 1.58
Combined	15.74 ± 1.90	16.14 ±1.54
Crown-rump length (mm)
Male	84.1 ± 3.7	84.9 ± 4.0
Female	82.7 ± 3.7	83.4 ± 3.0
Combined	82.8 ± 3.7	84.1 ± 3.5
Beak length (mm)
Male	13.2 ± 0.9	13.3 ± 0.5
Female	13.1 ± 0.6	12.9 ± 0.6
Combined	13.1 ± 0.7	13.1 ± 0.6
Percentage that were abnormal survivors	4.8	3.3

Validity criteria fulfilled:

not applicable

Conclusions:

The application of paraffin mixture on the shell of Mallard duck egg has no effect on embryo development.

Executive summary:

Paraffin mixture constitued of pentdecane, hexadecane, heptadecane, octadecane, nonadecane, 2,2,4,6,6-pentamethylheptane, 2,2,4,4,6,8,8-heptamethylnonane, 2,6,10,14-tetramethylpentadecane, and decahydronaphthalene, mixed in equal proportions, was applied on Mallard duck eggshell (5µL/egg). Embryonic survival, teratogenic potential and growth were evaluated.

No effect of oil application was noted, neither on embryo survival nor development.

Reference 4

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No guideline. The effects on embryonic development are investigated through direct application of the substance on the egg shell. A great number of eggs (100 per dose) were tested, which contributes to make the results statistically valid.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Oil is applied on the surface of fertile eggs. Embryonic survival time, teratogenic potential and growth are evaluated.

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No surrogate test material

Dose method:

other: application onto egg shell

Analytical monitoring:

no

Vehicle:

no

Details on preparation and analysis of diet:

No diet

Test organisms (species):

other: Gallus domesticus

Details on test organisms:

TEST ORGANISM
- Common name: Chicken
- Source: Commercial hatcheries: Whistling WIngs, Hanover, Ill., and Truslow Farms, Chestertown, Md.
- Age at test initiation (mean and range, SD): Eggs (48hr of development)

Limit test:

no

Total exposure duration (if not single dose):

15 d

Post exposure observation period:

No post-exposure period

No. of animals per sex per dose and/or stage:

100 eggs per dose. For sex ratio, see results.

Control animals:

yes, concurrent no treatment

Nominal and measured doses / concentrations:

5µL per egg

Details on test conditions:

SUBSTANCE APPLICATION:
5µL of paraffin mixture were applied by microliter pipet to the shell surface just below the air space of upright eggs (48 hrs of development) and permitted to spread freely


NO. OF EGGS
- For negative control: 100
- For treated: 100


TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: 37.5°C
- Relative humidity (%): 60

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: daily
- Remarks: all eggs were candled daily to determine mortality, and dead embryos were removed and examined.


EMBRYONIC WEIGHT
- Time schedule for examinations: All remaining eggs were opened on the 18th day of development and the embryos were removed and weighed.
- Remarks:


PATHOLOGY
- One half of the embryos were defeathered and placed in Bouin's solution for fixation and subsequent examination of soft tissues by the sectioning technique of Wilson (1965) and by dissection.
- The other half were cleared and staiend with alizarine red S according to the method described by Karnofsky (1965) for squeletal examination.



OTHER:
When removed from the shell, sex-ratios, crown-rump lengths, and beak lengths were recorded.

Details on reproductive parameters:

The following parameters were examined per egg:
- Embryos viable
- Clinical signs of toxicity, abnormalities and mortality


OTHER:
- Embryonic growth: embryo weight, crown-rump length, beak length
- Sex ratio

Reference substance (positive control):

no

Duration (if not single dose):

15 d

Dose descriptor:

other: NOELR

Effect level:

5 other: µL/egg

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Remarks:

Embryonic mortality

Duration (if not single dose):

15 d

Dose descriptor:

other: NOELR

Effect level:

5 other: µL/egg

Conc. / dose based on:

test mat.

Basis for effect:

reproductive parameters

Remarks:

Embryo weight

Duration (if not single dose):

15 d

Dose descriptor:

other: NOELR

Effect level:

5 other: µL/egg

Conc. / dose based on:

test mat.

Basis for effect:

reproductive parameters

Remarks:

Sex ratio

Duration (if not single dose):

15 d

Dose descriptor:

other: NOELR

Effect level:

5 other: µL/egg

Conc. / dose based on:

test mat.

Basis for effect:

other: Development: crown-rump length

Duration (if not single dose):

15 d

Dose descriptor:

other: NOELR

Effect level:

5 other: µL/egg

Conc. / dose based on:

test mat.

Basis for effect:

other: Development: beak length

Repellency factors (if applicable):

Not applicable

Mortality and sub-lethal effects:

For details on mortality and sub-lethal effects see Table no.1.

Effects on reproduction:

Effect on embryo development : see table1

Results with reference substance (positive control):

No reference substance

Further details on results:

No other details

Reported statistics and error estimates:

Embryonic weights, crown-rump lengths, and beak lengths were compared by one-way analysis of variance and Duncan's multiple range test. Survival data, sex distribution and numbers of embryos with one or more defects were compared by squared-chi analysis.
No statistical difference ws found between treated and control eggs for any invetigated parameter.

Table 1. Effects of paraffin mixture (5 µL/egg) on chicken embryos

 

	Control	Paraffin
Number treated	100	100
Percentage survival (Days 2 -15)	98	96
Sex ratio, M:F	54:46	50:50
Embryonic Weight (g)
Male	15.40 ± 1.24	14.85 ± 1.19
Female	15.11 ± 1.18	15.00 ± 1.15
Combined	15.25 ± 1.22	14.94 ±1.17
Crown-rump length (mm)
Male	74.4 ± 1.62	74.1 ± 1.55
Female	73.8 ± 1.65	73.7 ± 2.12
Combined	74.1 ± 1.63	73.9 ± 1.85
Beak length (mm)
Male	11.2 ± 0.49	11.0 ± 0.49
Female	11.1 ± 0.36	10.9 ± 0.37
Combined	11.2 ± 0.43	11.0 ± 0.42
Percentage that were abnormal survivors	3.5	5.8

 

Validity criteria fulfilled:

not applicable

Conclusions:

The application of paraffin mixture on the shell of chicken eggs has no effect on embryo development.

Executive summary:

Paraffin mixture constitued of pentdecane, hexadecane, heptadecane, octadecane, nonadecane, 2,2,4,6,6-pentamethylheptane, 2,2,4,4,6,8,8-heptamethylnonane, 2,6,10,14-tetramethylpentadecane, and decahydronaphthalene, mixed in equal proportions, was applied on chicken eggshell (5µL/egg). Embryonic survival, teratogenic potential and growth were evaluated.

No effect of oil application was noted, neither on embryo survival nor development.

Description of key information

There is no data available for this substance. However, key data is available for structural analogues. The data is read across to this substance based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

C10-C19 Paraffin mix presented a 26-week NOELR for Mallard ducks of 10000 mg/kg diet. No long-term toxicity of paraffin was observed neither to adult Mallard ducks, neither to their eggs.

C10-C19 Paraffin mix presented a 7-month NOELR for Mallard duck of 100000 mg/kg diet.

Paraffin mix (constitued of pentdecane, hexadecane, heptadecane, octadecane, nonadecane, 2,2,4,6,6-pentamethylheptane, 2,2,4,4,6,8,8-heptamethylnonane, 2,6,10,14-tetramethylpentadecane, and decahydronaphthalene, mixed in equal proportions) presented a 18-d NOELR for Mallard duck eggs of 5 µL/egg. The application of paraffin mixture on the shell of Mallard duck egg had no effect on embryo development.

Paraffin mix (constitued of pentdecane, hexadecane, heptadecane, octadecane, nonadecane, 2,2,4,6,6-pentamethylheptane, 2,2,4,4,6,8,8-heptamethylnonane, 2,6,10,14-tetramethylpentadecane, and decahydronaphthalene, mixed in equal proportions) presented a 15-d NOELR for chicken eggs of 5 µL/egg. The application of paraffin mixture on the shell of chicken eggs had no effect on embryo development.

Additional information

Several studies investigate the effects of a mixture containing equal concentrations of C₁₃, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉ n-paraffins, C₁₂, C₁₆, C₁₉ isoparaffins and C₁₀ cycloparaffin on mallard duck and chicken. In a first study (Hoffman, 1978), the mixture was applied directly on the egg shell (5 µL in one application) of mallard duck and chicken for 18 and 15 days respectively: the mixture had no effect on embryo development, or on sex ratio. Dietary studies with mallard ducks fed a diet containing 1% (i.e. 10 g/Kg diet) of the same paraffin mixture showed that the paraffin mixture had no effect on body and organ weights, reproduction, histopathology, hematology and serology (Patton, and Dieter, 1980; Coon and Dieter, 1981).